Immunohistochemistry UNIT 21.

4
1 1
Florence M. Hofman and Clive R. Taylor
1
University of Southern California, Los Angeles, California

ABSTRACT
This unit describes several methods for localizing specific antigens in various tissue and
cell preparations using immunohistochemistry (IHC). Protocols describe preparation of
suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells,
either freshly isolated or derived from suspension or adherent cultures; or fixed, paraffin-
embedded tissue sections. By careful selection of reagents, it is possible to detect two
or even three antigens simultaneously. For antigens that are sensitive to fixative, it may
be necessary to unmask the antigen by the antigen-retrieval technique. If there is cross-
reactivity between the secondary antibody and antigens present in the target cells or
tissue, the secondary antibody can be preabsorbed. Several new, sensitive amplification
techniques are currently available. The different IHC protocols are represented schemat-
ically and summarized in a table that also lists advantages and disadvantages of each
approach. Causes of background staining and ways to eliminate it are also discussed.
Curr. Protoc. Immunol. 103:21.4.1-21.4.26.  C 2013 by John Wiley & Sons, Inc.

Keywords: immunohistochemistry r antigen retrieval r double staining r staining fixed

tissues r staining frozen tissues r staining cell cultures r immunochemistry

INTRODUCTION
This unit describes several methods for localizing specific antigens in various tissue and
cell preparations using immunohistochemistry (IHC). Suitable material for IHC includes
fresh, unfixed, frozen tissue specimens (see Basic Protocol); unfixed cells, either freshly
isolated or derived from suspension or adherent cultures (see Alternate Protocol 1); or
fixed, paraffin-embedded tissue sections (see Alternate Protocol 2).

Support Protocol 1 describes “antigen retrieval,” a technique that is especially useful for
antigens that have been “masked” by fixation. If there is cross-reactivity between the
secondary antibody and antigens present in the target cells or tissue, the secondary anti-
body can be preabsorbed; Support Protocol 2 provides a method that enables polyclonal
secondary antibodies to be absorbed with mouse serum to eliminate cross-reactivity.
Cross-reactivity of the secondary antibody is usually a problem only in staining rat or
mouse tissue, because these animals are the most common animal sources of MAbs. It
is particularly useful when monoclonal antibodies (MAbs) generated in rats are used
on mouse tissue or MAbs generated in mice are used on rat tissue. Support Protocol 3
provides a strategy to help reduce background immunofluorescence staining, a problem
that is relevant for both frozen and formalin fixed sections.

By careful selection of reagents, it is possible to detect two or more antigens simultane-

ously (see Alternate Protocol 3).

Success of immunostaining depends on a number of factors: the source, specificity,

and quality of the primary antibody, and whether it is monoclonal or polyclonal; the
condition of the specimen; the fixation procedure; and the antigen-detection strategy.
These components are highly interdependent (see Critical Parameters). The different IHC
protocols are represented schematically in Figure 21.4.1 and summarized in Table 21.4.1,

Microscopy

Current Protocols in Immunology 21.4.21-21.4.26, November 2013 21.4.1

Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/0471142735.im2104s103 Supplement 103
Copyright C 2013 John Wiley & Sons, Inc.
 A

primary Ab labeled with enzyme

secondary Ab labeled with enzyme

treatment with biotinylated primary Ab

followed by the addition of avidin-enzyme
complex

treatment with biotinylated secondary

Ab followed by addition of biotin-avidin
complex with the enzyme bound through
the biotin

Key
primary Ab secondary Ab enzyme-substrate complex
avidin
biotin-avidin complex
antigenic determinants biotin
on specimen
avidin-enzyme complex

Figure 21.4.1 Diagrammatic illustration of (A) direct, (B) indirect, and (C) three-step immuno-
histochemistry.

which also lists advantages and disadvantages of each approach. With each additional
step there is an increase in sensitivity, but unfortunately this is offset by a possible
increase in nonspecific background staining. Causes of background staining and ways to
eliminate it are discussed in the Commentary (see Critical Parameters).

BASIC IMMUNOHISTOCHEMISTRY FOR FROZEN TISSUE SECTIONS

PROTOCOL
The following is a typical immunohistochemistry (IHC) protocol for frozen sections of
tissues from human and rodent sources; any frozen solid tissues can be used, including,
spleen, thymus, and lymph nodes. Cells or tissues from other species may be studied
Immuno- using appropriate immunological reagents. Essentially, staining is detected by sequen-
histochemistry
tial applications of a specific primary antibody and one or more reagents that amplify
21.4.2
Supplement 103 Current Protocols in Immunology
Table 21.4.1 Summary of Basic Immunohistochemistry Protocols

Protocol Uses Disadvantages

Direct
Enzyme-conjugated primary When primary antibody is Requires significant
antibody produced in the same species quantities of primary
as tissue to be stained; for antibody to directly label with
double staining; when the enzyme. Gives weak
secondary antibody produces staining because of minimal
high background amplification.
Two-step
Primary antibody + When quantity of primary May demonstrate increased
enzyme-conjugated antibody is limited; when the background due to secondary
secondary antibody or direct protocol does not antibody
Primary antibody + produce sufficient signal and
Polymer-conjugated with further amplification is
secondary antibody and HRP required.
Three-step
Primary antibody + For increased signal May produce increased
biotinylated secondary amplification background due to
antibody + avidin-conjugated endogenous biotin
biotinylated enzyme complex

the signal and are ultimately linked to an enzyme. In the presence of the appropriate
substrate, the enzyme causes precipitation of a colored reaction product at the site of
the antigen. This protocol is written for a horseradish peroxidase (HRP)–based detection
system; however other enzymes and colored substrate combinations may be used (see
Table 21.4.2).
Materials
Tissue, freshly isolated
PBS, pH 7.4 (APPENDIX 2A)
OCT embedding compound (Miles Labs)
Liquid nitrogen
Poly-L-lysine-coated slides (see recipe; also commercially available)
Acetone (reagent grade) or 4% (w/v) paraformaldehyde/PBS
0.3% (v/v) H2 O2 /PBS
Blocking solution: 5% (v/v) goat serum or 1% (w/v) BSA in PBS
Primary antibody diluted in either PBS, 1% (v/v) goat serum/PBS, or 0.1% (v/v)
saponin/PBS
Amplification system:
Biotin-avidin system, used for over 30 years because of its sensitivity,
convenience, and economic value. As such the biotin-avidin protocol has
become the yardstick for measuring reactivity in the research setting. To
use the protocol below with the biotin-avidin system, a biotinylated
secondary antibody directed against the species from which the primary
antibody was derived is required (Hsu et al., 1981).
Polymer-based detection system (available from multiple vendors), currently
preferred over earlier biotin-based methods, especially for automated
systems (Taylor and Shi, 2013; Fig. 21.4.2). The polymer-based
technology is useful because of lack of reactivity with endogenous biotin,
and also greater stability and sensitivity. Polymer-based systems are
Microscopy
available both for HRP and alkaline phosphatase (AP), as a second
21.4.3
Current Protocols in Immunology Supplement 103
 Table 21.4.2 Commonly Used Immunocytochemistry Enzymes and Substrates

Enzyme Substrate; color Advantages Disadvantages

Horseradish 3,3 -Diaminobenzidine Intense color; Endogenous peroxidase
peroxidase (DAB); brown permanent activity
3-Amino-9-ethyl Intense color; Endogenous peroxidase
carbazole (AEC); red contrasts well with activity
blue for double
staining
True Blue; blue Greatly increased Most effective for fixed
sensitivity tissues only
4-Chloro-1-naphthol Color contrast Soluble in alcohol
(4-CN); blue-black
Vina green; green Color contrast Soluble in alcohol
Alkaline Nitroblue tetrazolium Intense color Endogenous alkaline
phosphatase (NBT) and 5-bromo-4- phosphatase activity
chloro-3- indoyl
phosphate (BCIP); blue
Blue Substrate; blue Less intense color; Endogenous alkaline
but better for double phosphatase activity
staining
Fast red TR Red; red Color contrast for Endogenous alkaline
double staining phosphates activity
Glucose oxidase Nitroblue tetrazolium No endogenous Low staining intensity
(NBT); blue enzyme activity (high concentration of
primary and secondary
antibodies required for
effectiveness)

step 1
primary antibody

antigen

step 2
polymer containing
secondary antibody and enzyme

key: primary antibody secondary antibody

enzyme polymer, antibody, enzyme complex

Figure 21.4.2 Diagrammatic illustration of the two-step polymer staining technique.

Immuno-
histochemistry

21.4.4
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 enzyme for differential color staining, including double stains. To use this
protocol with a polymer-based detection system, a polymer-conjugated
secondary antibody/HRP complex is required.
HRP-conjugated avidin-biotin complex (ABC): e.g., ABC Elite (for use with
human tissue) or ABC Standard (for use with mouse tissue; both from Vector
Labs
Substrate (Table 21.4.1):
Diaminobenzidine (DAB) is prepared according to manufacturer’s
instructions (several variants exist including proprietary formats)
A number of additional substrates yielding differently colored reaction
products are available, including 3-amino 9-ethyl carbazole (AEC) (red),
4-CN blue-black, Vina green for HRP, and Fast Red for alkaline
phosphatase (AP)
Hematoxylin counterstain (Mayer’s; Sigma)
Aquamount (Thermo Scientific Shandon)
Petri dishes
Aluminum foil, cut into 3.5 × 3.5–cm pieces and labeled on the outside using a
waterproof marker
Cryostat
Coplin jar or staining dish with slide rack
Pap pen (Research Products International or Thermo Scientific Shandon)
Staining chamber (Fig. 21.4.3A)
Glass coverslips
Clear nail polish (optional)
Freeze the tissue sample
1. Place the freshly isolated tissue in a petri dish containing PBS and cut into the
desired number of approximately 0.5-cm3 pieces.
2. Place one tissue piece into the center of a labeled, precut piece of aluminum
foil.
3. Apply OCT embedding compound to the fragment in sufficient quantity to com-
pletely cover the tissue. Fold the foil to create a secure envelope.
OCT embedding compound is used to prevent dehydration at the edges of the sample
and to provide greater surface area for manipulation of the frozen tissue.

4. Drop the foil envelope directly into liquid nitrogen and allow the specimen to
remain there for 5 to 10 min. Subsequently, remove the aluminum envelope with
forceps and store at −70° or −120°C (for long-term storage).
Cryosection the tissue
5. To prepare the tissue for cryosectioning, bring the aluminum foil envelope contain-
ing the tissue to the cryostat on dry ice (preferable to liquid nitrogen), and open the
envelope inside the cryostat.
The cryostat should always be at approximately −20°C and, like a refrigerator, should
not be turned off except for cleaning. The tissue should be left in the cryostat for about
10 to 15 min to bring the tissue to −20°C, for easier cutting.

6. Cover the cryostat chuck with a thin layer of OCT embedding compound.
Place the tissue on the OCT-coated chuck in the appropriate orientation. Allow
the tissue/OCT/chuck complex to freeze solid (10 min at −20°C) inside the
cryostat.
Microscopy

21.4.5
Current Protocols in Immunology Supplement 103
 A

6 cm

glass slide

cm
30
2 cm
45 cm

B tissue sections

frosted wax pen outline

Figure 21.4.3 (A) Staining chamber. (B) Arrangement of sections on slide.

Covering the cryostat chuck is particularly important for such tissues as eye or brain
where orientation is critical. The temperature should be between −18° and −20°C; any
colder or warmer makes cutting the tissue difficult.

7. Set the cryostat to cut 5- to 8-µm-thick sections.

If the tissue contains experimental cells that are particularly large (e.g., brain), 8- to
10-µm-thick sections should be cut.

8. As the ribbon of tissue comes off the frozen block, place a poly-L-lysine-coated
slide under the sections and collect a maximum of three sections clustered together
on each slide (Fig. 21.4.3B).
Due to the temperature differential, as the section touches the slide, the section will
flatten and stick to the slide.

9. Allow the glass slide with the cryostat sections to air dry, preferably overnight at
room temperature.
Dried slides are ready for fixation.

Fix sections
Immuno- 10. Immerse the slides for 5 min in a Coplin jar or staining dish containing reagent-
histochemistry
grade acetone. Allow the slides to air dry for 10 min at room temperature.
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 As a fixation procedure, acetone should always be tried first, because acetone pro-
vides optimal retention of antigenic determinants and thus maximal staining. However,
acetone fixation often distorts morphology, depending on the specific tissue used. If
tissue morphology is critical, paraformaldehyde fixation can be used. Fixation for 10
min in 4% paraformaldehyde/PBS is an alternative, more gentle procedure that has
been shown to preserve some degree of antigenicity, although not as well as acetone.
Paraformaldehyde, however, produces superior morphology (see Critical Parameters).
Coplin jars can be used for fixing and washing up to five slides; a staining dish with
a slide rack can be used for up to 20 slides. These containers are useful because they
expose the slides to a large volume of solution that completely covers the slides.

11. Using a Pap pen, outline the tissue sections on the glass slide (Fig. 21.4.3B).
Encircling the sections using a Pap pen (water-repellant wax) creates a boundary that
prevents the reagent from spreading over the entire surface of the slide. When the
sections are outlined in this manner, small quantities of reagent (50 µl) can be used with
minimal risk of drying.
Some antigens are not stable when stored, so it is recommended that slides be stained
as soon as possible after preparation and fixation to obtain optimal staining. If slides
must be stored, storage at −70°C is usually adequate. However, it may be necessary to
test slides over time to determine if and when a particular epitope loses its ability to
bind antibody.
Stain the tissue
12. Place the slide in a staining chamber (Fig. 21.4.3A) and add PBS to cover the slide.
Incubate 5 min at room temperature.
IMPORTANT NOTE: Take special precautions to prevent slides from drying during
the staining procedure; it is best to add reagents to no more than three to four slides
at a time. When in doubt about drying, cover the slides with more reagent. Also, line
the staining chamber with paper towels soaked in sterile water to maintain a moist
environment.
Use a squeeze bottle or pipet to add the PBS, but do not apply directly to the tissue,
because the force of the liquid could lift up or destroy the tissue.
Perform all incubation and washing procedures in the staining chamber. If it is necessary
to interrupt the protocol, the slides may remain in PBS at any wash step for up to 2 hr
without deleterious effects.

13. Remove the PBS by tipping the slide so liquid drains into the staining chamber.
14. Treat the slides with 0.3% H2 O2 /PBS for 5 min or until bubbling stops.
If bubbling has not stopped after 5 min, reapply H2 O2 /PBS for another 5 min and, if
necessary, repeat until bubbling stops.
This incubation is used to eliminate endogenous peroxidase activity; if the enzyme to be
used in amplification is not peroxidase, pretreatment with H2 O2 is not necessary. Simi-
larly, pretreatment with H2 O2 is not needed if the tissue has no endogenous peroxidase
activity. This can be determined by performing steps 22 and 23 of this protocol on the
test slide (this should take 20 min). If there is red precipitate on the slide (substrate
without antibody), then peroxide pretreatment is necessary.

15. Add blocking solution to the slide and incubate 15 min. Tilt the slide to remove the
solution. Before going to the next step (applying the antibody), remove as much of
the liquid as possible without touching the tissue or letting the section dry.
Remove excess liquid by blotting the edge of the slide on a paper towel so the antibody
will not be unnecessarily diluted when it is added to the slide.

16. Apply diluted primary antibody in sufficient quantity to cover the tissue within the Microscopy
area marked by the wax pen. Incubate 60 min at room temperature.
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Current Protocols in Immunology Supplement 103
 Depending on the specificity of the primary antibody, it may be necessary to optimize
working dilution (see Critical Parameters). Optimization may be performed using cell
suspensions (see Alternate Protocol 1).
For convenience, it is possible to incubate the slides with primary antibody overnight
(approximately 18 hr), at room temperature or at 4°C. For overnight staining, the work-
ing dilution needs to be re-evaluated and usually modified by increasing the dilution.
Using mouse monoclonal primary antibody on mouse tissues presents a major problem
(see Alternate Protocol 3). Several commercially available blocking kits have been
developed.

17. Wash the slide twice in PBS, each time for 10 min.
Amplify signal
18a. For biotin-avidin system: Apply diluted biotinylated secondary antibody to the
tissue within the wax outline. Incubate 30 min at room temperature.
The use of the biotin-avidin system is has been the gold standard—however, due to
decreased sensitivity, background in specific organs such as kidney and liver, and fewer
incubation steps, the popularity of this system has decreased . However, this method is
useful for a wide variety of primary antibodies. Furthermore, biotinylated secondary
antibody can be diluted to the optimal concentration, making it more economical.
Biotinylated antibodies that recognize mouse, rabbit, goat, and rat primary antibodies
are available.
The secondary antibody should recognize immunoglobulin (Ig) from the species that is
the source of primary antibody (e.g., biotinylated goat anti-mouse is used to recognize
a mouse monoclonal primary antibody). Secondary rodent-specific polyclonal anti-
sera may need to be preabsorbed against normal rodent serum to remove background
reactivity with rodent Ig antigens (see Support Protocol 2).
18b. For the polymer system: Apply diluted polymer-conjugated secondary antibody/
HRP complex to the tissue section for 30 min at room temperature.
The polymer-based amplification technique is currently used in many research and most
clinical laboratories. This method is more sensitive than the biotin-avidin system due
to the increased numbers of peroxidase enzymes and secondary antibody molecules
bound to this structure. Background is reduced because no biotin is involved. The
polymer system is a two-step technique since the secondary antibody and HRP are both
bound together. However the polymer reagent comes pre-diluted, and is therefore less
economical.
The polymer system is limited to use with rabbit and mouse primary antibodies.

19. Wash the slide twice in PBS, each time for 10 min.
Detect bound antibodies
20. For the avidin-biotin technique: Incubate the slides with avidin-biotin complex
(ABC) conjugated to HRP for 20 min.
21. Wash the slide twice in PBS, each time for 10 min.
22. Add DAB or AEC substrate solution to the slide and incubate 5 to 10 min. Check
the slide after 5 min for color development.
Always prepare the working substrate solution immediately before adding to the slide,
and add H2 O2 as the final ingredient. If this solution is not used within 5 min of
preparation, the staining intensity will be significantly diminished.
To evaluate the slide, place a white paper towel underneath the slide and look for brown
Immuno- or red color. If the slide looks dark, stop the reaction immediately by washing the slide
histochemistry in tap water. To examine the slide more closely, tilt it to remove most of the liquid, then

21.4.8
Supplement 103 Current Protocols in Immunology
 add a coverslip and examine the slide using a light microscope. If the positive control
slide is understained, reapply a freshly prepared substrate solution.

23. Transfer the slide to a Coplin jar or staining dish and wash 10 min in running
tap water. Direct the stream of water at a corner of the container to provide good
circulation and to avoid contact with the tissue on the slide.
Counterstain tissue
24. Immerse slide in hematoxylin counterstain for 30 sec to 2 min.
Hematoxylin is reusable and should be kept in a covered staining dish on the benchtop.
However, carryover of water from the slides into the hematoxylin will eventually dilute
the stain. Thus, after five to ten uses, the incubation time required will steadily increase.
When nuclear staining is no longer crisp and clear, the stain should be replaced with
fresh hematoxylin.

25. Wash the slide 10 min in tap water.

26. Place 1 to 2 drops of Aquamount on the tissue.
27. Place a coverslip over the tissue and, if desired, seal with clear nail polish to prevent
drying. Allow the nail polish to dry thoroughly before examining the slide under a
light microscope.
Sealed slides can be stored indefinitely.

IMMUNOHISTOCHEMICAL STAINING OF CULTURED CELLS ALTERNATE

PROTOCOL 1
Immunohistochemistry (IHC) of cells in culture is an excellent system to complement
studies on tissue sections. Tissues provide a static picture of a process, and cultured cells
can provide insights into active processes. Single-cell preparations from cultured cells
can be used to evaluate a variety of immunologic reagents, including those that recog-
nize cytokines and activation antigens as well as adhesion molecules. It is also easier to
identify the subcellular localization of particular antigens (e.g., nuclear, Golgi, or surface-
associated antigens) in single-cell preparations. In addition, IHC of cell preparations, as
with tissue sections, can be combined with in situ hybridization, in situ apoptosis, or
double immunostaining (see Alternate Protocol 3) to examine several parameters simul-
taneously. Use of cultured cells versus tissue sections often facilitates visual interpretation
of staining results when the multiple staining protocol (see Alternate Protocol 3) is being
performed; cell preparations should be used to optimize conditions for different proce-
dures, and may be used as a preliminary protocol for tissue staining. Cultured cells can
also be used as a positive control for determining the optimal concentration of antibody
for a transiently expressed antigen such as adhesion molecules (e.g., E-selectin). Cells in
culture are ideal for the study of transient antigens because these cells can be activated
and fixed at the appropriate time for optimal antigen expression. After the cells are in
a single-cell suspension, a cytospin centrifuge is used to prepare slides. Fixation and
staining follow the procedure described in the Basic Protocol. Adherent cells can also be
stained in situ.
Additional Materials (also see Basic Protocol)
Cultured cells in appropriate medium
Cell suspension medium: any isotonic balanced salt solution (e.g., RPMI) with 1%
to 2% (v/v) FBS or 1% (w/v) BSA
Complete medium with 10% (v/v) FBS (e.g., RPMI-10; APPENDIX 2A)
0.5 mM EDTA (APPENDIX 2A) or 0.5% (w/v) trypsin/0.5 mM EDTA

Microscopy

21.4.9
Current Protocols in Immunology Supplement 103
 Sorvall RT 6000 centrifuge and H-1000B rotor (or equivalent)
Cytospin centrifuge (Shandon/Lipshaw), including chambers and filters
Additional reagents for counting viable cells by trypan blue exclusion (APPENDIX 3B)
1. Culture cells in the appropriate medium under conditions appropriate for the exper-
iment.
Either suspension cells or adherent cells are suitable for IHC. Cells can be cultured in
any of a variety of tissue culture vessels—Petri dishes or flasks. Adherent cells can also
be cultured on sterile coverslips in dishes or in glass chambered slides.

2a. For adherent cells to be stained in situ: At the time appropriate for staining, wash
the cells twice with PBS, each time for 10 min. Air dry the slides overnight at room
temperature. Proceed with staining, beginning at step 7.

For cells grown in suspension cultures

2b. Harvest the cultures, count viable cells (APPENDIX 3B), and resuspend cells in cell
suspension medium at 0.5 × 106 viable cells/ml.
Cells should always be counted and percent viability determined using trypan blue
exclusion as described in APPENDIX 3B, because dead cells usually exhibit nonspecific
positive staining.
To harvest adherent cells, wash the cells twice in cold PBS. Incubate the cells briefly in
0.5% trypsin/0.5 mM EDTA or 0.5 mM EDTA (EDTA alone is preferred for studies of
cell-surface antigens because trypsin destroys many surface antigens). When the cells
have detached, add 2 to 3 vol complete medium that contains 10% FBS. Centrifuge the
cells 10 min at 200 × g (1000 rpm in H-1000B rotor), room temperature. Resuspend
the cells in a small (1- to 5-ml) amount of cell suspension medium and determine the
viable cell count. Adjust the volume of the cell suspension to give 0.5 × 106 viable
cells/ml.
To harvest suspension cells, transfer cells and medium to a centrifuge tube and centrifuge
10 min at 200 × g. Resuspend the cell pellet in a small amount of cell suspension medium
(usually 1 to 2 ml) and determine the viable cell count. Adjust the volume of the cell
suspension to give 0.5 × 106 viable cells/ml.

3b. Assemble the cytospin apparatus, consisting of slide, filter, and sample chamber.
4b. Carefully resuspend the cells and add 150 to 200 µl (105 cells) to the cytospin
chamber.
This cell number is appropriate for leukocytes; for larger cells such as endothelial cells,
fewer cells (5 × 104 cells/slide) should be used.
Cells should be added to the chambers as quickly as possible. If it takes >5 min to
fill all the chambers, the cells will settle and cluster in the funnels and deposit on the
slide as a densely packed crescent, rather than a single thin layer of cells in a circular
pattern. It may be difficult to interpret the results when cells are densely packed on the
slide.

5b. Centrifuge the cytospin assembly 5 min at 70 × g (800 rpm), room temperature.
6b. Remove the cytospin assembly, separate the slides from the filters, and allow the
slides to air dry overnight at room temperature.
Dried filters can be reused 2 or 3 times.

7. Fix and stain the slides (see Basic Protocol, steps 10 to 27).

Immuno-
histochemistry

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IMMUNOHISTOCHEMISTRY OF FIXED PARAFFIN-EMBEDDED ALTERNATE
SECTIONS PROTOCOL 2
Formalin- or paraformaldehyde-fixed tissue retains excellent morphology; however, the
antigen of interest may have been denatured or altered during the fixation process such
that recognition by primary antibody is diminished or completely obliterated. This is true
for a wide range of antigens. This problem is addressed by the use of a combination of
recently developed reagents specific for fixed antigens, as well as the “antigen retrieval”
protocol for unmasking antigens (Table 21.4.3).

Table 21.4.3 Partial List of Antibodies that React with

Formalin-Fixed Human Cell Determinants

Antigens Sourcea
T cell
CD3 D, N
CD4 N
CD6 B
CD43 B, D, N, S
CD45 RO D, S
B cell
CD20 B, D, N, S
CD21 D, N
CD45 RA B, D, N
CD23 N
Plasma cell B, D
Other leukocytes
CD68 (macrophages) B, D, S
CD15 (granulocytes) D, N, S
CD57 (natural killer) B, D, N
CD45 (common leukocyte) B, D, S
Miscellaneous
CD31 (endothelial cells) B, D, S
CD34 (endothelial cells) B, N
CD35 (dendritic reticulum) D
HLA Dr B, D, S
ICAM-1 B
Myelin basic protein B, D
bcl2 B, D, S
p53 B, D, S
Ki-67 (proliferation) B, D, S
PCNA (proliferation) B, D, S
Cytomegalovirus D, N, S
Epstein-Barr Virus D, N
Human immunodeficiency virus D
a Commercial suppliers: B = BioGenex; D = Dako; N = Novocastra Lab; and
S = Signet Lab. See APPENDIX 5. Microscopy

21.4.11
Current Protocols in Immunology Supplement 103
 Additional Materials (also see Basic Protocol)
Tissue: fixed, ideally in 10% (v/v) neutral buffered formalin or 4% (w/v)
paraformaldehyde in PBS, pH 7.4 (APPENDIX 2A), paraffin-embedded, and
sectioned using a microtome (5 µm thickness).
Staining dishes containing 100%, 95%, and 80% ethanol
Xylenes
Histoclear
0.3% (v/v) H2 O2 /methanol
1. Immerse the tissue fragment in 10% neutral buffered formalin or 4% paraformalde-
hyde in PBS for 8 to 24 hr, depending on the size of tissue.
The optimal size for the tissue fragment is 5 × 5 × 3 mm. Tissue should not be left in
fixative for more than 48 hr. If tissue cannot be processed by 48 hr, then the tissue should
be transferred to 70% ethanol; the tissue can remain in this solution for several weeks,
If tissue is already embedded in paraffin, begin with step 4.

2. Process the tissue: Dehydrate 15 min in 80% ethanol; 15 and then 20 min in 95%
ethanol; three times, 20 min each, in 100% ethanol; and finally, three times, 20 min
each, in xylenes. Embed tissue in paraffin and cut sections 5- to 8-µm thick. Place on
poly-L-lysine-coated or SuperPlus slides (as described above).
Tissue processing and sectioning are standard procedures and usually performed in a
dedicated histology laboratory on a fee-for-service basis.

3. After the tissue sections are applied onto the slide, place slides in the oven for 1 hr at
60°C.
This drying procedure removes water from beneath the tissue section and makes the section
adhere better to the slide.

4. Deparaffinize the slides by placing them for two 5-min changes each in Histoclear,
100% ethanol, and 95% ethanol. Between changes, quickly blot the edge of the slide
on a paper towel to remove excess liquid, but do not allow the slide to dry.
Histoclear is a substitute for potentially toxic xylenes.
Immersion steps are performed using either Coplin jars or staining dishes with slide racks,
depending on the number of slides that are being processed. The containers are filled so
that the slides are completely immersed when they are inside the container. The solutions
are reusable; however, they must be replaced once or twice a week depending on the
number of slides processed and the amount of liquid carried over into the containers with
the slides. Usually, the solutions are changed after every 50 slides. However, because these
reagents absorb water from the atmosphere, they should be changed at least every 2 weeks
no matter how many slides have been treated.

5. To block endogenous peroxidase, incubate the slide 10 min in 0.3% H2 O2 /methanol.

This step should always be performed when peroxidase is the enzyme used for amplifica-
tion; it should be omitted when other enzymes (Table 21.4.2) are used for detection.

6. Wash slides twice in PBS, 10 min each at room temperature.

7. Proceed with immunohistochemistry (see Basic Protocol, steps 15 to 27).

ALTERNATE IMMUNOHISTOCHEMISTRY TO DETECT TWO ANTIGENS

PROTOCOL 3 SIMULTANEOUSLY
There are several features of immunohistochemistry that make it possible to use this
Immuno- methodology to detect two antigens simultaneously. This method is often referred to
histochemistry
as the double-staining method. Because this procedure uses the light microscope to
21.4.12
Supplement 103 Current Protocols in Immunology
 first primary antibody biotinylated first avidin-biotin-peroxidase
secondary antibody complex + substrate

second primary alkaline phosphatase- conjugated

antibody second secondary antibody + substrate
Key
tissue with two distinct biotin
antigens avidin
first first avidin/biotin
primary antibody secondary antibody
avidin/biotin
peroxidase +
second second substrate
primary antibody secondary antibody
alkaline phosphatase
+ substrate

Figure 21.4.4 Double-staining (see Alternate Protocol 3).

visualize the reaction product, it is possible to identify reaction products in the context
of tissue morphology and topography based on their different colors. The staining results
are relatively permanent. It is often possible to localize the antigens on a cellular or
subcellular level. There are a number of possible combinations of enzymes and substrates
(see Table 21.4.2).

Several factors should be considered in designing a double-staining experiment. The

relative cellular locations of the antigens to be stained are important. In sequential
staining, to reduce steric interference, intracytoplasmic or nuclear antigens should be
stained before surface antigens are labeled. The primary antibodies for the two different
antigens must be from different species. Ideally, one primary antibody could be a mouse
monoclonal antibody (MAb) and the second a rabbit polyclonal antibody. With this
arrangement, the two secondary antibodies would be specific for mouse Ig and rabbit Ig.
The final consideration is whether the substrate used in the first staining process alters or
diminishes the antigenicity of the second antigen.

The double-staining procedure is essentially two single-staining procedures performed

consecutively (Fig. 21.4.4). To obtain excellent color contrast, the double-staining
Microscopy
protocol described here uses alkaline phosphatase–conjugated secondary antibody and
21.4.13
Current Protocols in Immunology Supplement 103
 alkaline phosphatase blue substrate (Vector Labs), followed by horseradish peroxidase
(HRP)–conjugated secondary antibody with AEC substrate (red; see Basic Protocol,
steps 18 to 23). Other enzyme-substrate combinations are possible (see Table 21.4.2).
Alternatively, two ‘stains’ may be performed together if a mouse primary antibody is
used for one target antigen and a rabbit monoclonal antibody for the second. The de-
tection system then employs both mouse and rabbit linker systems simultaneously, each
with a separate chromogen. Rabbit monoclonal antibodies are widely available against a
growing range of antigens, making this the preferred method.

With automated staining platforms, many manufactures now offer ‘double staining’
kits, designed to run on their specific platforms. Using these kits greatly simplifies the
procedure, and increases the chances of a good outcome. However, if these kits are used,
the manufacturer’s instructions should be followed, although incubation times may be
adjusted for different research applications.
Additional Materials (also see Basic Protocol)
Slides with cryostat sections (see Basic Protocol, steps 1 to 9), tissue culture cells
(see Alternate Protocol 1, steps 1 to 6), or deparaffinized paraffin-embedded
sections (see Alternate Protocol 2, steps 1 to 6)
Primary antibody
Alkaline phosphatase–conjugated secondary antibody (Vector Labs)
0.125 M levamisole/H2 O
Alkaline phosphatase Blue Substrate (Vector Labs or Kirkegaard & Perry)
Staining chamber (Fig. 21.4.3) wrapped in foil
Stain with first antibody
1. Incubate slide of cryostat sections, cultured cells, or deparaffinized paraffin-
embedded sections 30 to 60 min in the primary antibody for the first antigen.
Include slides for appropriate controls (see Table 21.4.4). Wash slide twice in PBS,
each time for 10 min.
The primary antibody used here should be prepared in a different animal than that used
to prepare the antibody detected using the HRP detection system.

2. Wash slide twice in PBS, each time for 10 min.

3. Incubate slide 30 min in alkaline phosphatase–conjugated secondary antibody.
The alkaline phosphatase–conjugated secondary antibody should recognize Ig from the
species used to prepare the first primary antibody.

4. Wash slide twice in PBS, each time for 10 min.

5. Dilute 0.125 M levamisole 1:1 (v/v) in PBS. Incubate the slide 30 min in this
solution.
Levamisole blocks human alkaline phosphatase, which is present in a wide range of cell
types, but it does not inhibit bovine alkaline phosphatase, which is the enzyme conjugated
to the secondary antibody.

6. Prepare the alkaline phosphatase Blue Substrate according to the manufacturers’

instructions.
The substrate should not be allowed to stand >3 min before it is applied to the slide.
This substrate provides the best contrast to the red product produced by the horseradish
peroxidase reaction.

7. Remove the slide from the levamisole solution and add the substrate solution imme-
Immuno- diately to the slide. Incubate 45 to 60 min in a foil-wrapped slide chamber at room
histochemistry
temperature, examining the slide every 15 min for color development.
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Supplement 103 Current Protocols in Immunology
Table 21.4.4 Control Slides for the Double-Staining Protocol

Slide First staining Second staining Results

1. Experimental First primary antibody + Second primary antibody Blue and red
alkaline phosphatase– + biotinylated secondary staining
conjugated secondary antibody +
antibody + substrate HRP-conjugated ABC +
substrate
2. Control First primary antibody + Controla + biotinylated Blue staining only
alkaline secondary antibody +
phosphatase–conjugated HRP-conjugated ABC +
secondary antibody + substrate
substrate
3. Control Controla + alkaline Second primary antibody Red staining only
phosphatase–conjugated + biotinylated secondary
secondary antibody + antibody + HRP-
substrate conjugated ABC +
substrate
4. Control First primary antibody + PBS Blue staining only
alkaline
phosphatase–conjugated
secondary antibody +
substrate
5. Control PBS Second primary antibody Red staining only
+ biotinylated secondary
antibody + HRP-
conjugated ABC +
substrate
a Control: no serum, preimmune serum, or isotype-matched immunoglobulin.

The substrate should develop to a deep blue color within 45 to 60 min, depending on the
amount of antigen and primary antibody bound. The slide should be examined every 5
min to avoid high background.
This substrate is very light sensitive, so all incubations must be performed in an aluminum
foil–wrapped slide chamber.

8. Stop the reaction by rinsing with PBS. Wash twice, each time for 10 min in PBS.
Stain with second antibody
9. Stain slide using appropriate primary antibody for second antigen, biotinylated
secondary antibody, HRP-conjugated ABC, and AEC substrate (see Basic Protocol,
steps 16 to 23), but do not counterstain the slide.
10. Mount slide in Aquamount and add coverslip (see Basic Protocol, steps 26 to 27).

EXPOSING ANTIGENS USING THE ANTIGEN RETRIEVAL METHOD SUPPORT

PROTOCOL 1
Formalin or paraformaldehyde fixation cross-links protein, often making antigens in-
accessible to specific antibodies. The antigen retrieval protocol, which combines high
temperature with buffers at different pH, has been very effective in unmasking antigenic
determinants which may be masked in fixed tissue or fixed cell preparations, particularly
determinants within the nucleus or cytoplasm (Taylor and Shi, 2013). The method also
is known as HIER (Heat Induced Epitope Retrieval), umasking, or uncloaking. Frozen
Microscopy

21.4.15
Current Protocols in Immunology Supplement 103
 tissues or fresh cell preparations normally have well preserved and exposed antigens with
minimal protein cross-linking, and they cannot withstand the high temperature required
for antigen retrieval.

With the growing use of automated staining systems many leading manufacturers now
sell ‘antigen retrieval’ solutions that have been formulated and proven for use with their
respective systems. The use of these reagents should be considered in these circumstances;
they also perform effectively in manual protocols.
Materials
Slides prepared from fixed tissue or cultured cells (see Alternate Protocol 1, steps 1
to 5)
Sodium citrate buffer: 0.1 M citric acid/0.1 M sodium citrate, pH 6.0
PBS (APPENDIX 2A)
Microwave oven, standard model operating at a frequency of 2450 MHz with
highest power setting 600 W
Plastic Coplin jars with vented screw caps
1. Wash slide prepared from fixed tissue or cultured cells in distilled water twice, 5 min
each.
2. Dilute sodium citrate buffer to 1 mM and fill plastic Coplin jars three-quarters full.
3. Add slide to Coplin jar of sodium citrate buffer and loosely cover the jar with a vented
screw cap. Heat 5 min in a microwave oven set at the highest power setting.
4. Check the jar for fluid level and add distilled water if necessary to bring the level to
three-quarters full.
5. Heat the slide again in the microwave oven for 5 min at the highest power setting.
6. Remove the jar from the microwave oven and cool 15 min.
7. Wash the slides twice, 10 min each, in PBS.
The slide is now ready for immunohistochemistry (see Basic Protocol, steps 15 to 27).

SUPPORT ABSORPTION OF SECONDARY POLYCLONAL ANTISERA TO

PROTOCOL 2 ELIMINATE BACKGROUND
This absorption protocol is particularly useful when monoclonal antibodies (MAbs)
generated in rats are used on mouse tissue or MAbs generated in mice are used on rat
tissue. Nonspecific background is due to the cross-reactivity of secondary antibodies with
Ig epitopes in the tissue specimen. For example, a polyclonal anti-rat reagent used as a
secondary antibody may cross-react with naturally occurring mouse Ig epitopes in mouse
tissue, particularly in mouse hematopoietic organs. Therefore, the polyclonal secondary
antibody should be absorbed with mouse serum (Fig. 21.4.5). This cross-reactivity of
the secondary reagent is usually a problem only in staining rat or mouse tissue, because
these animals are the most common animal sources of MAbs.
Materials
Polyclonal secondary antibody: e.g., biotinylated goat anti–rat Ig or biotinylated
goat anti–mouse Ig
Normal serum from the same species as the tissue
Sorvall RT 6000 centrifuge and H-1000B rotor (or equivalent)

Immuno- 1. Add undiluted polyclonal secondary antibody to an equal volume of appropriate

histochemistry normal serum in a small microcentrifuge tube. Incubate 18 to 24 hr at 4°C.
21.4.16
Supplement 103 Current Protocols in Immunology
 mouse tissue

unabsorbed biotinylated unabsorbed goat anti-rat

polyclonal goat anti-rat antibody cross-reacts with
antibody with a mixture mouse Ig epitopes (and rat
of specificities primary antbody)

mouse
serum

mouse tissue
absorption removes goat absorbed goat anti-rat
anti-rat antibody that antibody binds specifically
cross-reacts with mouse to rat MAb
Ig epitopes
Key
biotinylated goat biotinylated goat primary monoclonal
anti-rat antibody anti-rat antibody rat anti-mouse
that cross-reacts antibody
with mouse Ig
epitopes

mouse tissue mouse Ig epitopes

Figure 21.4.5 Absorption of cross-reactive antibodies to epitopes in mouse tissue from sec-
ondary reagents (see Support Protocol 2).

2. Centrifuge the mixture 10 min at 800 × g (2000 rpm in H-1000B rotor), room
temperature.
3. Remove the supernatant and use at the appropriate dilution for the secondary reagent.

METHOD FOR REDUCING BACKGROUND STAINING SUPPORT

PROTOCOL 3
The ability to stain mouse tissues is important to the researcher. As mentioned previously,
many primary antibodies are prepared as mouse monoclonal reagents. Thus, using any
anti-mouse secondary antibody on mouse tissue will result in unacceptable levels of
background. This is particularly relevant when staining immune organs, which express
high levels of Ig. This problem is important for both frozen and formalin-fixed sections,
but to a lesser extent for cell preparations because cells can be washed to remove exoge-
nous Ig. There are several approaches to reducing background Ig staining. The method
Microscopy
suggested below uses a proprietary blocking reagent (M.O.M. Mouse Ig Blocking;
21.4.17
Current Protocols in Immunology Supplement 103
 Vector Laboratories) and biotinylated anti-mouse Ig as the secondary antibody. There
are several approaches to reducing background Ig staining. The method suggested below
uses biotinylation of the primary antibody before using the reagent. This technique has
been somewhat successful if the manufacturer’s instructions are faithfully followed. The
streptavidin-HRP reagent is used as the detection mechanism.
Additional Materials (also see Basic Protocol)
Mouse monoclonal primary antibody (e.g., Dako)
M.O.M. mouse immunodetection kit (Vector Laboratories, cat. no. MKB-2213)
Horseradish peroxidase–conjugated streptavidin (streptavidin-HRP; Life
Technologies, cat. no. S0911)
1. Follow steps 1 to 15 of the Basic Protocol.
2. Add mouse blocking reagent to the slide according to the manufacturer’s instructions.
3. Add mouse monoclonal primary antibody at the appropriate concentration and incu-
bate for 30 min at room temperature.
4. Wash twice with PBS, each time for 10 min.
5. Add the secondary biotinylated anti-mouse Ig (from M.O.M. kit; follow manufac-
turer’s instructions), and incubate for 30 min.
6. Wash twice with PBS, each time for 10 min.
7. Add enough streptavidin-HRP to the cover the specimen on the slide and incubate for
30 min at room temperature.
8. Wash twice with PBS, each time for 10 min.
9. Continue with Basic Protocol, steps 21 to 27

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see APPENDIX 5.

Poly-L-lysine-coated slides
Clean glass slides in 70% ethanol and air dry. Dip the slides individually into poly-
L-lysine solution (Sigma) diluted 1:10 in water for 5 min. Dry the slides >30 min in
a 55°C oven. Store at 4°C and use within 1 week.

COMMENTARY
Background Information possible to examine fluorescence patterns and
It has been almost 40 years since enzyme- cell and tissue morphology simultaneously.
labeled antibodies were first introduced and IHC utilizes a light microscope for visual-
used to identify specific antigens in rou- ization, so antigen localization and cell and
tinely processed tissues (Taylor, 1974). This tissue morphology can be observed at the
technique, immunohistochemistry (IHC), is same time. Third, fluorochrome-based signals
methodologically identical to immunofluo- last only 3 to 10 days, depending on the signal
rescence (IF), which has been in use since intensity and storage conditions. The colored
1941 (Coons et al., 1941). The increasing precipitate in IHC staining, particularly with
popularity of IHC over IF is due to four main 3,3 -diaminobenzidine (DAB; Table 21.4.2),
factors. First, IF requires special equipment, remains vibrant for years and provides
a fluorescence microscope, and a designated an excellent permanent record. Fourth, IF
darkened area. IHC, on the other hand, can be fluorochromes are light sensitive, so special
Immuno- performed using a standard light microscope. care must be taken throughout the staining
histochemistry Second, IF requires a dark field, so it is not procedure to minimize loss of signal. The
21.4.18
Supplement 103 Current Protocols in Immunology
enzymes and most substrates used in IHC, on Primary antibody
the other hand, are relatively light insensitive, The critical issue in IHC is always the
and therefore the staining procedure can easily specificity of the primary antibody. Therefore,
be performed on the open benchtop without whenever possible, MAbs should be used.
any unusual precautions. The disadvantage of Often, however, due to the lack of availabil-
some IHC chromogens is their known carcino- ity or the necessity for double staining, this
genicity, but using gloves when handling these is not always possible. The recent availability
reagents is an adequate safety precaution. The of a growing range of rabbit monoclonal anti-
availability of a variety of enzyme-conjugated bodies has greatly improved the situation, in-
antibodies and colored substrates (see Ta- cluding the ability to perform double stains, as
ble 21.4.2) has made IHC more convenient noted previously. Staining human tissue with
and accessible to many laboratories. rabbit polyclonal antibodies often yields sub-
IHC procedures are continually being stantial background, because rabbit Ig appears
modified to accommodate new experimental to bind more readily to human Fc receptors
situations. In the past, most specimens than do sera from other species, but this is not
processed for IHC were fixed with formalin, an issue for rabbit monoclonals. If an effective
and the antibodies used for detecting antigens mouse or rabbit monoclonal antibody is not
were polyclonal reagents made primarily in available, one option is to use a polyclonal an-
rabbits. These polyclonal antibodies identified tibody prepared in a species other than rabbit;
a wide range of epitopes and were specifically goat and horse sera have been shown empir-
selected for binding to epitopes that remained ically to give the lowest background levels.
resistant to the fixation procedures. Although However, if only rabbit primary antibodies are
these polyclonal antibodies had a broad range available, then blocking with 5% (v/v) normal
of specificities, they frequently gave high goat serum is useful in reducing background.
levels of background staining. The advent of If nonspecific staining persists, further dilu-
MAbs brought with it a vast improvement tion of the rabbit primary antibody combined
in the specificity of staining along with a with an increase in the concentration of sec-
relative elimination of background staining. ondary reagent is often effective. To determine
This made it possible to detect such diverse whether the staining observed with the primary
elements as membrane receptors, secreted ex- antibody is significant, normal serum from the
tracellular products, and signal-transduction animal source of the primary polyclonal an-
enzymes. Previously, the disadvantage of us- tibody, or an isotype-matched Ig for MAbs,
ing MAbs was that these reagents proved to be should be used at a concentration equivalent to
useful only in analyzing fresh, acetone-fixed that used in the experimental staining. The nor-
specimens, and were not particularly effective mal serum or isotype-matched control slides
with formalin-fixed specimens because many should produce no staining.
of the epitopes detected by MAbs are formalin The most common primary antibodies cur-
sensitive. This problem is being addressed rently in use are MAbs generated from mouse
by new methods that include enhanced hybridoma or rabbit cells. Mouse monoclonal
detection of relatively low levels of antigen antibodies work well on non-murine tissue,
and selective development of MAbs that but are not usually useful for staining mouse
recognize formalin-resistant epitopes. These specimens because the secondary antibody
techniques have greatly expanded the useful- must necessarily be an anti–mouse Ig, and this
ness of IHC in a wide range of experimental results in high background staining in mouse
situations. tissue, particularly in hematopoietic tissue and
in areas adjacent to blood vessels. Therefore,
Critical Parameters and monoclonal or polyclonal antibodies from rab-
Troubleshooting bit are recommended when staining mouse tis-
Immunohistochemistry (IHC), like other sue, even with tissues that are relatively im-
antibody-based techniques, requires careful munologically sequestered, such as brain. An
selection and titration of antibody reagents alternative approach to staining mouse tissue
and use of appropriate controls. Linscott’s with a mouse primary MAb is to conjugate
Directory of Immunological and Biological it directly with an enzyme (e.g., horseradish
Reagents provides a complete, regularly up- peroxidase, HRP) or linking reagent (e.g., bi-
dated listing of commercially available anti- otin; see Table 21.4.1). Mouse MAb may be
bodies, reagents, and kits. In addition, fixation used on certain mouse cell lines (e.g., fibrob-
and the method chosen for signal amplification lasts and T cell lines) because these cells
Microscopy
can affect the results. do not normally have Ig on their surface.
21.4.19
Current Protocols in Immunology Supplement 103
 Nevertheless, when using mouse MAb on Controls
mouse cells, it is always necessary to in- Proper controls must always be included
clude a control slide that has been exposed to with each staining procedure. Either preim-
mouse serum or isotype-matched Ig in place of mune serum or isotype-matched Ig should be
the primary antibody and subsequently treated used as a negative control to ensure that the
identically. Such a control slide determines staining observed is not due to background;
the extent of nonspecific background bind- a positive control slide should always be in-
ing of the anti-mouse secondary antibody and cluded in each staining experiment to verify
subsequent reagents. Mouse tissue specimens the activity of all the reagents.
can, however, be successfully stained with rat In staining tissue, the specificity of the pri-
MAbs, which are becoming more common. mary antibody is critical and should be eval-
The most common secondary antibody for rat uated on both positive and negative control
MAbs is goat anti–rat Ig. In some tissues, tissues and cell preparations. To test reagents
particularly mouse hematopoietic organs, this that supposedly detect antigenic markers ap-
secondary reagent can cause background stain- pearing only on activated cells, known posi-
ing because of the cross-reactivity between tively responding cells should be stimulated
rat Ig and mouse Ig. To identify nonspecific and harvested at a time point that is ap-
background in these experiments, use a con- propriate in terms of response kinetics. Un-
trol slide in which PBS is substituted for the stimulated or nonresponding cells should be
MAb during primary antibody incubation. If cultured and harvested in parallel. Then, cyto-
a diffuse positive reaction is observed, the centrifuge preparations of the cells should be
secondary antibody should be absorbed with stained with the antibody in question. For ex-
mouse Ig to remove cross-reactive components ample, to test the primary antibody for the ad-
(see Support Protocol 2). When mouse MAbs hesion molecule E-selectin, endothelial cells
are used to stain rat tissue, the secondary goat should be treated for 3 to 4 hr with an appropri-
anti-mouse reagent should be absorbed with ate reagent (e.g., TNF or IL-1) to up-regulate
rat serum. With the proper precautions and expression of the antigen. Both stimulated
controls, MAbs can be used on rodent tissues and unstimulated cell preparations should be
to obtain consistent, clear, and reliable results. stained. Unstimulated cells should be rela-
tively negative for E-selectin (<2% positive),
and stimulated cells should be >50% posi-
Antibody titration tive, suggesting that the primary antibody is
The optimal concentration of primary an- specific.
tibody should be determined using a standard In general, activated human cell lines and
checkerboard titration experiment, taking into peripheral blood leukocytes provide good
consideration the manufacturers’ recommen- sources of positive controls for the titration
dations and the source of the reagent. For of many primary reagents. Activated mouse
MAbs derived from hybridoma supernatants spleen cells are usually a good source of pos-
using undiluted supernatant, 3- and 10-fold itive control murine cells. Untreated control
dilutions are good starting points. For MAbs and stimulated cell preparations should be
derived from ascites fluid, dilutions of 1:10, fixed with acetone and stored at −70°C for
1:100, and 1:300 are more appropriate. Un- future use in reagent evaluation and titration.
less instructed otherwise by the manufacturer, It is important to control for endogenous
dilutions of 1:200 for biotinylated horse peroxidase activity when HRP is used as
anti–mouse Ig and 1:400 for biotinylated goat the detection enzyme, especially in tissues
anti–rabbit Ig are optimal. For polyclonal anti- displaying high numbers of macrophages or
bodies, dilutions ranging from 1:100 to 1:1000 granulocytes, as in inflammation. A simple
should be tested. Titration experiments should preliminary test for assessing the extent of
always include appropriate controls—e.g., an endogenous peroxidase is to hydrate a fixed
irrelevant antibody or preimmune serum in slide with PBS, apply only the substrate (e.g.,
place of a primary polyclonal antibody or an AEC), and incubate for the appropriate reac-
isotype-matched Ig in place of primary MAb. tion time (e.g., 10 min). The slides are then
Ideally, these controls should give no back- washed in water and examined for evidence of
ground staining at the optimal concentration. staining; there should be none. If there is reac-
The final working concentration for primary tivity, H2 O2 pretreatment should be performed
antibodies used on cytocentrifuge preparations to eliminate endogenous peroxidase activity,
Immuno- is usually 2- to 3-fold higher than for the same and then the optimized pretreatment should be
histochemistry
primary antibody on tissue sections. performed on all experimental slides.
21.4.20
Supplement 103 Current Protocols in Immunology
 The double-staining technique requires line phosphatase as the precipitating enzyme.
several controls to ensure accurate interpreta- If DAB is used for IHC, alkaline phosphatase
tion of results. The control slides are described substrate (Vector Labs) is recommended as the
in Table 21.4.4. Slide 1 is for the experimental second chromogen. For these experiments, the
procedure, and should always be performed same controls as described for double IHC (Ta-
in duplicate, with at least two specimens per ble 21.4.4) are recommended to ensure proper
slide. Slides 2 through 5 are control slides for evaluation of results.
various steps in the procedure. Slide 2 should
show staining equivalent to that of slide 4; this Fixation
demonstrates that the second staining proce- Once an antibody that recognizes a par-
dure does not give background (nonspecific ticular antigenic determinant based on other
red color) and that no antigen is lost during the techniques (e.g., immunoblot analysis) is iden-
long staining procedure with the use of dif- tified, the next step is to take a known pos-
ferent reagents. Slide 3 should be equivalent itive control cell preparation and fix it us-
to slide 5; this shows that there is no non- ing acetone. Acetone is a gentle fixative that
specific blue staining from the first protocol causes minimal antigenic denaturation, mem-
and that the first staining procedure did not brane permeabilization that is limited but suf-
destroy, alter, or diminish the antigenicity of ficient to allow antibodies to enter the cell,
the determinant recognized by the second pri- and fixation of the tissue that is sufficient for
mary antibody. Use of alkaline phosphatase some degree of morphological identification.
with a blue substrate followed by peroxidase Acetone also appears to be effective for local-
with AEC has provided the best results. ization of specific cell surface as well as cyto-
In double staining, the order of antigens plasmic antigens. In the author’s experience,
to be labeled should also be carefully consid- acetone is the fixative of choice for determin-
ered. Ideally, cytoplasmic antigens should be ing optimal antibody concentrations of the vast
stained first and surface antigens stained sec- majority of both monoclonal and polyclonal
ond. The reverse approach may cause steric primary antibodies.
interference. Optimal conditions for each anti- Occasionally, acetone-fixed cells known to
gen should first be determined using a single- be positive for a given antigen fail to yield a
staining protocol. If the antigens in question positive result. If acetone-fixed positive con-
are both cell-surface determinants, there may trols show no staining when, for example,
be some degree of interference in double- these control cells are positive based on other
staining experiments. This can be assessed by criteria—e.g., mRNA analysis, immunoblot
examining control slides for single staining; as analysis, or ELISA—the time interval between
mentioned previously; there should be approx- preparing the slides and staining may have
imately the same number of positive cells on been too long. For example, IFN–γ is a partic-
slide 4 as on slide 2 and on slide 5 as on slide ularly sensitive antigen and must be stained
3. The visible color is also an indicator of po- within 24 hr after slide preparation. Other
tential overlap in double staining: if the stains cytokines (e.g., IL-6) are more stable and can
appear bright red or blue, there is no overlap be detected by IHC after slides have been
in staining, but if the color is purple, there is stored 1 or 2 weeks at room temperature.
significant overlap. If the results of a double- Another possible cause for negative results
staining experiment demonstrate dual-staining using a known positive control is that the
cells (e.g., staining with anti-CD4 and anti- antigen in question (e.g., intracellular cy-
IFN-γ), another set of slides where the known tokine) may have leaked out of the cell during
outcome is single-staining cells (e.g., staining washing steps because acetone fixation did
with anti-macrophage and anti-IFN-γ) should not adequately cross-link the cell membrane.
be double stained. By comparing the results of In such cases, it is worthwhile to use 4%
the two experiments, the individual patterns in (w/v) paraformaldehyde/PBS, pH 7.4, another
the dual-staining cells should be obvious. gentle fixative that has been shown to preserve
For best results, when double-staining is some antigenic determinants (e.g., IL-6,
used for IHC and in situ hybridization or in but not CD4 or CD8). Paraformaldehyde
situ apoptosis, it is advisable to fix the tis- fixation cross-links proteins, often sufficiently
sue, continue with staining, and then begin the to protect cytoplasmic antigens from being
second procedure. If results with AEC (red) washed out of the cell. Electron microscope
are too faint or undetectable, substitute DAB (EM)–grade paraformaldehyde does not
(brown) for the substrate. In situ hybridiza- appear to have a significant advantage over
Microscopy
tion or apoptosis use either peroxidase or alka- the non-EM grade. If positive controls fixed
21.4.21
Current Protocols in Immunology Supplement 103
 with paraformaldehyde continue to show 1 mM sodium citrate buffer, pH 6.0, but 1 mM
no staining, the antibody solution may be Tris·Cl and 1 mM EDTA at pH 8.0 have also
supplemented with 0.1% (w/v) saponin or been used. In general, higher-pH solutions are
0.1% (v/v) Triton X-100 (final concentration). more effective for intensifying staining, but
Because these detergents dissolve lipids, using care should be used because they may cause
them enhances membrane permeabilization the tissue to disintegrate and fall off the slide
and removes lipids that may also obscure or occasionally produce nonspecific staining
intracellular antigenic determinants. Saponin of the nucleus. Studies have shown that even
or Triton X-100 should also be present in the using distilled water (pH 6.0) on slides at high
PBS washes between applications of primary temperatures enhances staining. The critical
and secondary antibodies, as this eliminates high-temperature step is most often performed
or decreases nonspecific background. Saponin in a microwave oven at the highest power set-
should be prepared as a 10% (w/v) stock ting. High temperature can also be produced
solution, and diluted in PBS on the day of by autoclaving 10 min at 120°C, heating 2 min
staining. in a domestic pressure cooker at an operating
Proteinase K treatment after fixation has pressure of 103 kPa/15 lb/in.2 , or heating 20
also been used to increase access to antigens, to 40 min in a 100°C water bath. Each antigen
especially nuclear antigens. The enzyme is may require different conditions, so heating
usually added to the slides before application conditions and buffer pH may need to be
of the primary antibody. However, this pro- optimized. Antigen retrieval is most effective
cedure is rather harsh, and requires careful for cytoplasmic determinants; however, some
calibration of treatment duration versus pro- enhancement of surface receptors has been ob-
teinase K concentration. First, try incubating served (Shi et al., 1995). Additionally, antigen
the slide 10 min in 10 µg/ml proteinase K (di- retrieval has provided limited but substantial
luted in PBS) at room temperature. If the tissue access to antigens in formalin-fixed archival
comes off the slide or background is high, first tissue.
decrease the duration of the incubation, then
decrease the proteinase K concentration. Signal amplification
A problem that often occurs in formalin- Once the primary antibody titer has been
fixed tissues is extensive denaturation of pro- established and the proper fixation procedure
teins. To determine whether the antibody of in- identified, optimal conditions for maximizing
terest can be used following formalin fixation, the signal must be identified. There are several
one fresh positive sample (cells or cryostat sec- ways to intensify staining without increasing
tions) is fixed with acetone and another is fixed the concentration of primary antibody. The
with formalin for 10 min. Both slides are duration of the primary antibody incubation
washed twice in PBS and subjected to IHC (see can be increased from 30 to 60 min, or
Basic Protocol, steps 13 to 27). The staining even overnight at room temperature. For an
intensities of both slides are compared to de- overnight incubation, it is necessary to take
termine the effect of formalin fixation, if any. additional steps to prevent slides from drying
Because formalin fixation affects many anti- out. Placing a paper towel saturated with ster-
gens and because formalin-fixed tissues are ile water in the bottom of the staining chamber
widely used, many companies have made an is usually sufficient. If the slide has dried at
effort to identify and manufacture antibodies any point during the procedure, it will exhibit
to specific antigenic epitopes that are resistant different kinds of nonspecific staining; e.g.,
to formalin fixation, and these antibodies can a pink to dark red haze over the entire tissue
be used specifically for formalin-fixed tissue (with AEC substrate), a granular deposit over
(see Table 21.4.3). the entire tissue, intense staining of nuclei,
In addition to these new reagents, the or staining around the edges of the tissue.
antigen retrieval method has become widely Staining intensity may also be increased by
used to “unmask” denatured or covered incubating the slides 30 min at 37°C in an
antigens resulting from fixation. This method incubator or commercially available slide
described in Support Protocol 1 uses a com- warmer.
bination of high temperature and buffers of Another possible source of background
different pH to retrieve or unmask the antigen is the peroxidase enzyme. Peroxidase is a
in formalin- or paraformaldehyde-fixed tissue naturally occurring enzyme that is found
(Shi et al., 1991). High temperature is used in macrophages and granulocytes. Endoge-
Immuno- to reverse cross-linking of proteins. The nous peroxidase activity can be eliminated
histochemistry
buffer most commonly used in this process is simply by a brief incubation in H2 O2 ,
21.4.22
Supplement 103 Current Protocols in Immunology
Table 21.4.5 Troubleshooting Guide for Immunohistochemistry

Problem Possible reason Solutions

Tissue has “lace-like” Tissue was frozen improperly Exercise greater care in
network appearance (e.g., too slowly), not coated freezing (see Basic Protocol,
well with OCT, or frozen and steps 2-4)
thawed
Focusing on a single cell Tissue section is too thick Recut sections thinner
layer is impossible and/or (10 µm); check the
hematoxylin is too dark thickness designation on
cryostat, it may be inaccurate
Positive cells are detected Endogenous peroxidase Try longer or multiple H2 O2
after incubation with only the activity is present treatments; if endogenous
substrate (negative control) peroxidase background
persists (e.g., in bone
marrow), use another enzyme
(e.g., alkaline phosphatase)
Tissue sections come off the Slides were not properly Retreat the same slides or use
slide during treatment coated with poly-L-lysine commercially prepared
precoated microscope slides
(e.g., Superfrost/Plus Slides,
Fisher)
In cytocentrifuge preparation, Cell suspension remained in Prepare fewer slides at one
the cells have accumulated in the cytocentrifuge chamber time
a crescent formation, all to too long
one side
Entire tissue is brightly Slide has dried at some point Discard slides; avoid
staining during the procedure allowing the slide to dry
Edge of tissue is staining Several reagents have Increase washing time after
intensely accumulated under the tissue secondary antibody and ABC
to 30 min
Staining with AEC is Too much substrate has Reduce primary antibody
brownish-yellow instead of precipitated at the antibody concentration 50%, until
red site AEC staining appears red
For a series of slides cut at This antigenic determinant is For consistently satisfactory
the same time, slides stained unstable and may have been results, perform staining on
immediately are intensely altered because the sections sections or cytospin
positive; slides stained within were left at room temperature preparations within 24 hr of
2 or 3 days are weak or for too long fixation; if storage is
negative necessary, −70°C is usually
adequate
Staining for a particular Primary antibody was Primary antibody should be
antigen appears weaker than improperly handled—e.g., the re-titrated
usual on the positive control reagent was left out on the
slide bench for a long time or
inadvertently frozen and
thawed repeatedly
There is little or no staining Primary antibody may be Use only sterile tips in
for the antigen in question, contaminated handling all reagents and/or
and there appears to be add 0.1% sodium azide to
precipitate on the slide antibodies, but do not use in
cell culture studies; discard
antibody if contamination is
confirmed Microscopy

Continued 21.4.23
Current Protocols in Immunology Supplement 103
 Table 21.4.5 Troubleshooting Guide for Immunohistochemistry, continued

Problem Possible reason Solutions

Faint background is seen all Blocking was insufficient Block with 5% goat serum
over slide before primary antibody is
applied, and dilute primary
antibody in 1% goat serum
Background staining is seen Absorption of secondary Absorb secondary antibody
around all blood vessels in antibody was insufficient with mouse serum overnight
mouse tissue at 4°C (Support Protocol 2)
Staining is seen in the Secondary antibody is Use 0.1% Triton X-100 in
absence of primary antibody producing background PBS during the washes after
addition of secondary
antibody
Red particles are seen on AEC is precipitating in stock Prepare new AEC stock
tissue solution solution
Red (AEC) color intensity is There is too little H2 O2 in the Prepare new working AEC
fainter than usual on positive AEC; too much liquid on the solution and treat again
control slide when AEC was added
Slides fade immediately after Mounting medium contains Change to alcohol-free
adding coverslip alcohol, which solubilizes mounting medium or use
AEC DAB as substrate
Bubbles are seen on the slide Coverslip was not placed on Remove coverslip by placing
the slide slowly slide in 37°C water until the
coverslip slides off, then
carefully add a fresh coverslip
(Note: do not remove
coverslip manually as tissues
will come off)

leaving insignificantly low levels of back- Although both alkaline phosphatase and glu-
ground reactivity. cose oxidase produce less intense signals than
The substrates for peroxidase are com- peroxidase, these enzyme labels are used for
monly 3-amino-9-ethyl carbazole (AEC), double-staining experiments (Alternate Proto-
which gives a bright red precipitate, and 3,3 - col 3). A wide range of substrates that produce
diaminobenzidine (DAB), which results in an precipitates of different colors (e.g., black,
intense brown precipitate. The DAB signal can blue, red, purple) are available (Vector Labs,
be intensified further with a nickel sulfate solu- Dako) to produce specific contrasting signals.
tion. A new commercially available substrate Colloidal gold–labeled secondary antibod-
for peroxidase, TrueBlue, from KPL increases ies, which are used for immunogold staining,
the sensitivity of staining by 20- to 50-fold are very sensitive and produce minimal back-
and provides a bright blue reaction product. ground. With the availability of smaller (1-
However, the pH of the substrate solution is and 5-nm) particles, these conjugates have be-
rather acidic (pH 6.0), so it works best on come more useful (De Waele et al., 1991).
paraffin-embedded, formalin-fixed tissue; it is However, silver enhancement is generally nec-
not suitable for frozen sections or cell prepara- essary to maximize contrast. The one advan-
tions. The peroxidase reaction develops into an tage of the immunogold method is that each
intensely colored precipitate with a very low black dot theoretically represents a single anti-
rate of diffusion, thereby producing crisp stain- genic determinant; therefore, this technique
ing. The signal is long lasting, often vivid for is potentially more quantitative than enzy-
6 months to several years. Alkaline phos- matic methods, which depend on the concen-
phatase or glucose oxidase may also be used tration and duration of enzyme activity. The
Immuno-
histochemistry as the detection enzyme (see Table 21.4.2). granularity of the staining product makes it

21.4.24
Supplement 103 Current Protocols in Immunology
Figure 21.4.6 Immunohistochemistry of virus-infected mouse liver tissue. Tissue was stained
with rabbit anti–lymphocytic choriomeningitis virus (LCMV) antibody using alkaline phosphatase
substrate (blue) and mouse monoclonal anti-CD4 using peroxidase substrate (red) without coun-
terstaining. Arrows indicate single positive cells with no overlap in staining. Magnification is 100×.
For the color version of this figure, go to http://www/currentprotocols.com/protocol/im2104.

difficult to interpret the results, and methods termined by counting the number of positive
for signal quantitation are still crude. For elec- and negative cells and in tissue preparations by
tron microscopy, immunogold is an excellent counting the number of positive cells per unit
technique. area. However, IHC is not suitable for deter-
For guidance concerning the problems most mining the actual number of molecules provid-
often encountered in IHC experiments and ing the specific signal observed in either tissue
methods for troubleshooting and resolving or cell preparations. With the constant devel-
them, see Table 21.4.5. opment of new reagents and protocols, the ver-
satility and usefulness of this procedure should
Anticipated Results increase. Figures 21.4.6 and 21.4.7 show re-
The goal of an immunohistochemistry sults of double-staining experiments.
(IHC) experiment is to obtain clearly defined
staining in the context of tissue morphology
that will be reproducible and remain readable Time Considerations
for years. There have been several attempts Both cryostat sections and cytocentrifuge
to address the sensitivity of IHC. The various cell preparations require an overnight drying
staining systems have been compared (Elias step before fixation. The optimal three-step
et al., 1989), and the results indicate that the protocol (Basic Protocol) requires anywhere
sensitivity of the primary antibody is the pri- from 4 hr to 24 hr, depending on the length of
mary critical parameter. With this in mind, it time needed for primary antibody incubation.
is not advisable to use IHC as a method for Double staining usually takes between a mini-
quantifying the amount of antigen at the cellu- mum of 8 hr and a maximum of 24 hr because
lar level, but rather, it should be used as a tech- it is not advisable to incubate overnight with
nique for demonstrating the absence or pres- both primary antibodies. Thus, starting from
ence of an antigen and its distribution. Percent fresh tissue or cells, the entire procedure can
Microscopy
positive cells in cell preparations can be de- be completed within 48 hr.
21.4.25
Current Protocols in Immunology Supplement 103
 Figure 21.4.7 Immunohistochemistry of brain tissue derived from animals with experimental
allergic encephalomyelitis. Tissue was stained with rabbit anti-nitrotyrosine using alkaline phos-
phatase substrate (blue) and mouse monoclonal anti-CD11 using peroxidase substrate (red)
without counterstaining. Arrows indicate positive double staining for a nitrotyrosine subpopula-
tion of CD11-positive cells. Magnification is 400×. For the color version of this figure, go to
http://www/currentprotocols.com/protocol/im2104.

Literature Cited Shi, S.R., Imam, A., Young, L., Cote, R.J.,
Coons, A.H., Creech, H.J., and Jones, R.N. 1941. and Taylor, C.R. 1995. Antigen retrieval im-
Immunological properties of an antibody con- munohistochemistry under the influence of pH
taining a fluorescent group. Proc. Soc. Exp. Biol. using monoclonal antibodies. J. Histochem.
Med. 47:200-202. Cytochem. 43:193-201.
De Waele, M., Renmans, W., Segers, E., Jochmans, Taylor, C.R. 1974. The nature of Reed-Sternberg
K., Salmon, I., Depardieu, C., Dehou, M.-F., cells and other malignant cells. Lancet 2:802-
and Van Camp, B. 1991. Leukemia and lym- 807.
phoma immunophenotyping in cell smears with Taylor, C.R. and Shi, S-R. 2013. Techniques
immunogold-silver staining. Am. J. Clin. Pathol. of Immunohistochemistry: Principles, pitfalls
96:351-359. and standardization. In Comprehensive Di-
Elias, J.M., Margiotta, M., and Gabore, D. 1989. agnostic Immunohistochemistry, Fourth edi-
A comparison of the peroxidase-anti-peroxidase tion (D.J. Dabbs, ed.) pp. 1-40 Elsevier,
(PAP), avidin-biotin complex (ABC), and la- New York.
beled avidin-biotin complex (LAB) methods for
detection of glucagon in paraffin-embedded hu-
man pancreas. Am. J. Clin. Pathol. 92:62-67.
Key References
Hsu, S.M., Raine, L., and Fanger, H. 1981. The Taylor and Shi, 2013. See above.
use of avidin-biotin peroxidase complex (ABC) An in-depth historical background analysis and
in immunoperoxidase technique: A comparison critical overviews of theoretical and practical as-
between ABC and unlabeled antibody (PAP) pects of immunohistochemistry techniques, with ap-
procedures. J. Histochem. Cytochem. 29:577- pendix listings of commercial sources for antibod-
580. ies, reagents, and equipment.
Shi, S.R., Key, M.E., and Kalra, K.L. 1991. Antigen This is an excellent chapter discussing the latest
retrieval in formalin-fixed, paraffin-embedded advances in immunohistohemistry techniques, for
tissues: An enhancement method for immuno- the laboratory investigator and clinical laboratory.
histochemical staining based on microwave The development of the protocols and troubleshoot-
oven heating tissue sections. J. Histochem. Cy- ing analyses are very useful for those planning to
Immuno- tochem. 39:741-748. establish these methodologies.
histochemistry

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Supplement 103 Current Protocols in Immunology