Multiple Antigen Immunostaining Procedures

Abstract

Detection of multiple antigens in the same tissue section can be done by combining a range of immunohisto/cytochemical techniques based either on light microscopic chromogenic precipitates or fluorochrome labeling. Light microscopic techniques preferred for this purpose use combinations of immunogold silver staining (black precipitate), immunoperoxidase, immunoalkaline phosphatase and immunogalactosidase methods using chromogens of different colors. Fluorochrome labels favored for these combinations include AMCA (blue), FITC (green), rhodamine (orange-red) and Cy5 (far red), their matching synthetic members from the Alexa series, or quantum dots. Antibodies directly labeled or those from noncross-reacting animal species (e.g., mouse, rabbit, goat, guinea pig etc.) can be applied simultaneously. When the antigens of interest are in separate cells or cell compartments (e.g., in cell membrane, cytoplasm or nucleus), and only cross-reacting antibodies are available, there have also been ways of avoiding unwanted cross-talk. These include the exploitation of the shielding effect of chromogens; inactivation of immuno-sequences of the first staining by using either acidic elution, formaldehyde fixation or microwave heating; combining unlabeled and hapten-labeled antibodies; or using labeled monovalent F(ab) secondary antibodies. In this chapter we briefly discuss the principle of multiple antigen immunolabeling and provide useful protocols for its performance.

1. Introduction

The immunohistological demonstration of multiple antigens in a single tissue section has many applications and is commonly used to elucidate the topographic relationships of antigenically defined cell populations, and to correlate phenotypic information with functional or prognostic markers, or with microbial infection (1). In addition, single or double label immunohistochemistry may be combined with in situ hybridization to identify genes or mRNAs, and translated protein side-by-side (2–4). The combination of these techniques depends primarily on the compatibility of the antigen retrieval techniques required for the individual antigens, and the stability of the RNA and DNA sequences in question (4).

Two basic strategies have evolved for the detection of multiple antigens in a tissue section. One strategy is to combine immunoenzymatic methods that use different chromogenic-substrate reactions (5–7) (see Chapter 24) and/or the silver-enhanced immunogold technique (IGSS) (8–10) (see Chapter 30). The different colored reaction products can easily be studied against the recognizable background structure with a conventional light microscope, particularly when traditional histological stains such as hematoxylin for nuclei or PAS for basement membranes are additionally applied. With careful balancing of the subsequent chromogenic reactions, up to four antigens situated in separate cell populations or within different compartments of the same cell (nucleus, cytoplasm, or cell membrane) can be distinguished in a single tissue section (7, 10).

The second strategy uses combinations of different antibodies detected with fluorochromes of distinct emission maxima (at least 40 nm difference) recognized as discernible colors (11, 12). The visualization of fluorochromes requires the use of fluorescence or confocal laser scanning microscopes with special filters. The combinations of noncross-reacting immunoreagents labeled with distinct fluorochromes allow for the clear demonstration of overlapping fluorescence signals of merged colors characteristic of antigen coexpression in the same tissue/cell compartment (13, 14). With proper fluorochrome and filter combinations up to four antigens can be selectively immunostained side by side in the same tissue sections (11).

Multiple antigen staining protocols can be further subdivided depending on whether antibodies of one immunoreaction can interact with those used in the subsequent immunoreaction(s). When such “cross-talk” between the multiple staining reactions can be excluded (e.g., when mouse and rabbit antibodies are combined in a dual labeling experiment), the immunoreagents of identical layers (e.g., primary antibodies or link/detection antibodies) may be mixed and used simultaneously. If crosstalk cannot be avoided in the experimental design (i.e., when two monoclonal mouse primary antibodies of the same subclass are used), the reaction steps for each antigen have to be completed consecutively, including some blocking steps. In this chapter we present guidelines and describe protocols suitable for performing immunohistochemical double and triple antigen detection. Although we have not detailed specific protocols for double/multiple staining using immunofluorescence, the principles of staining are identical, and suggestions for immunofluorescent antigen detection are provided.

1.1. Double/multiple Antigen Immunostaining

1.1.1. Principles of Reagent Combination

The principles followed for multiple antigen staining are identical to those for single antigen staining. The primary difference is that one must prevent unwanted cross-reactions (cross-talk) between the different staining sequences. Thus, the combination of primary and secondary antibodies is a major determinant of the appropriate double/multiple antigen detection protocol. Crosstalk is not expected when the primary antibodies originate from different species such as mouse, rabbit, goat, sheep, guinea pig or monkey (7, 11), the secondary antibodies are species-specific and purified from cross-reacting elements, and the chromogen-substrate reactions are distinct. The same is true for combinations consisting of antibodies of different immunoglobulin isotypes (i.e., IgG and IgM), or subclasses (i.e., IgG₁ and IgG₂) that are detected with isotype/subclass-restricted secondary antibodies (15). Cross-talk will also not occur if the primary antibodies are of identical species or isotype and are directly coupled to different enzymes or fluorochromes (4, 16). Another method to reduce cross-talk is to apply preformed complexes of the primary antibodies and their cognate enzyme or fluorochrome coupled secondary reagents (17, 18). This approach, however, can be laborious as one must first determine the optimal saturation for the complexes or eliminate uncomplexed reagents prior to performing the immunoreaction.

In all of the above combinations, reagents of identical layers (primary and secondary) may be applied simultaneously in the same incubation steps. Only the chromogenic-enzyme reactions, if relevant, need to be carried out consecutively, (e.g., alkaline phosphatase activity revealed first followed by peroxidase development).

1.1.2. Combining Immunofluorescence Techniques

Immunofluorescence methods can be combined only if cross-talk between the immunological sequences can be excluded, regardless of whether the antigens are situated in the same or different cells/compartments. Fluorochrome molecules are small, usually below the molecular range of 1 kDa, and thus, unlike chromogenic precipitates (discussed below), they cannot mask antigenic epitopes to prevent cross-reacting secondary detection reagents from binding to primary antibodies of prior reaction layers (12). On the other hand, immunofluorescence methods are ideal for co-localization studies at the light microscopic level, where mixed colors of intermediate emission wavelength indicate two antigens in the same structure (12, 13). Fluorochrome labeling is also the method of choice for double fluorescence in situ hybridization (FISH) and has gained widespread use for detecting chromosomal abnormalities in diagnostic pathology (19). The Alexa series of recently designed fluorochromes represent a pH- and relatively photo-stable alternative to the traditional fluorescing dyes with significantly brighter light emission at the same wavelength (12). The fluorochromes most commonly used for combined antigen detection and their absorption and emission maxima and fluorescent color are summarized in Table 1. More recently fluorescent semiconductor nanoparticles called quantum dots (Q-dot® nanocrystals, Invitrogen, Molecular Probes, Carlsbad, CA) have been used for dual antigen detection in tissue sections (20). These cadmium-based nanoparticles can be coupled to a variety of secondary detection reagents, including anti-immunoglobulins, streptavidin, and protein A. Q-dots® are available in multiple emission spectra (a function of their size and composition), have very intense and narrow emission characteristics, and are highly stable over time, eliminating several of the major problems of dye-based fluorescent detection technologies.

Table 1.

The most common fluorochromes used for multiple antigen immunostaining

Fluorochrome	Alexa® series	Abs. max. (nm)	Emiss. max. (nm)	Color	References
AMCA (7-aminomethyl-4-methylcoumarin-3-acetic acid)	Fluor 350	350/346	450/445	Blue	(12, 13, 34, 38)
DAPIa (4′6-diamidino-2-phenylindole, dihydrochloride)		348	461	Blue	(12)
FITC (fluorescein-5-isothiocyanate)	Fluor 488	494	518	Yellow-green	(12, 13, 18, 34, 38, 39)
TRITC (tetramethyrhodamine-5-isothiocyanate)	Fluor 546	545/556	580/573	Orange-red	(12)
Cy3 (cyanin 3)		550	570	Orange-red	(13, 38, 39)
Texas Red®	Fluor 594	590	615	Red	(12, 13, 18, 34)
7-AADa (7-aminoactinomycine D)		546	647	Red
Cy5 (cyanin 5)	Fluor 647	650	670	Far red	(18)

^aDNA/nuclear stains

Significant improvements in microscopic resolution and color contrast can be achieved when the fluorescence signals are studied with confocal laser scanning microscopy and the optical layers generated are digitally merged, magnified and the color channels are assigned to artificial colors using commercially available software programs (Fig. 1). In this way, the cellular distribution of objects as small as 0.2–0.5 µm such as desmosomes and gap junctions can be analyzed in known tissue volumes of identical sections, well beyond the resolution of the light microscope (14). The same principle of analyzing the two signals in separate recording channels is utilized in double epi-illumination microscopy (epipolarization for gold-silver and epi-fluorescence for Fast Red) (21), or when CCD camera-captured individual images either of immunofluorescence of immunoenzyme nature are enhanced, filtered and then merged using imaging software (22, 23).

Fig. 1.

Double immunofluorescence detection of cytoplasmic cytokeratin 19 intermediate filaments (CK19) (FITC, green/filamentous) and the cell-membrane associated connexin 43 (Cx43) gap junction protein (Cy3, red/dot-like) in the stratified squamous epithelium of human cervix. Combination of mouse monoclonal (CK19) and rabbit polyclonal (Cx43) antibodies and distinct fluorochrome-labeled secondary antibodis. Confocal laser scanning microscopy, a projection of five optical layers.

1.1.3. Combining Immunostains Based on Catalytic Chromogenic Precipitation

Noncross-reacting antibodies, however, are not always available in the laboratory. Methods based on catalytic chromogenic precipitation, including immunoenzyme techniques and immunogold silver staining (IGSS), can be used for combinations of either noncross-reacting or cross-reacting immune sequences, provided the antigens to be detected are in separate cells/compartments (6). In cases, however, when cross-talk is expected, it is essential that the immunostains are performed sequentially and certain precautions are taken.

Preventing Cross-Talk Based on the Shielding Effect of Chromogenic Precipitates

In enzyme reactions, catalytic precipitates will be formed as long as the enzyme is active, and has not been concealed by the accumulating precipitate. In the IGSS method although autocatalysis is continuous, the appearance of silver grains in the developer solution limits further intensification. In both cases catalytic precipitates form permanent shells shielding the immunological sequences used, which can be exploited to avoid potential but unwanted cross-talk in multiple antien detection protocols. The silver shell of IGSS and the compact 3, 3′-diaminobenzidine tetra-hydrochloride (DAB) precipitate can completely block the immunological sequences of the associated antigen/antibody complex (10). Therefore, when the antigens of interest are expected in different cell populations or in different cellular compartments, primary antibodies of the same animal species may be used in consecutive immunoreactions. IGSS (black) or immunoperoxidase (IPO)/DAB (brown) should be used to reveal the first immunoreaction, followed by the application of either IPO/3-amino-9-ethylcarbazole (AEC; red), immunoalkaline phosphatase (IAP)/Fast Blue (blue), nitro-blue-tetrazolium (NBT; purple-black), Fast Red (magenta/red), or New Fuchsine (magenta/red) to demonstrate additional antigen(s) (8–10, 24) (see Chapter 24).

DAB is probably the most versatile chromogen in immunohis-tochemistry. Its brown color can be modified when used in the presence of nickel-chloride (purple-black), cobalt-chloride (dark-blue) or copper sulfate (dark brown) (25). Also, DAB and its metallic complexes can be further contrasted to form a black precipitate with silver intensification (26). Immunoreactions resulting in electron-dense precipitates, such as immunogold, IGSS, and IPO/DAB-complexes, have also been used widely for double/multiple antigen detection at the ultrastructural level (1, 27). For light microscopy, one of our preferred double labeling techniques combines two IPO/DAB detections, whereby the first enzyme label is developed with DAB-Ni to form a purple-black precipitate that provides excellent contrast with the light-brown DAB “only” precipitate used for the second immunodetection (Fig. 2). IPO techniques used with different chromogenic reactions (e.g., DAB-Ni, DAB and V-VIP) may allow clear distinction of as many as three antigens in the same section (28). Chromogenic precipitates, most commonly used for combined antigen immunodetection, are summarized in Table 2.

Fig. 2.

Double staining of membrane and nuclear antigens using consecutive immunoreactions. (a) Depicts a large B-cell lymphoma (LBCL) doubly stained for the B-cell-associated membrane antigen CD20 and the B-cell-associated nuclear antigen B-cell-specific activator protein (BSAP). Note the colocalization of BSAP and CD20 in the tumor cells. (b) Depicts the same LBCL doubly stained for BSAP and the membrane-associated T-cell-associated antigen CD3 (arrows). Note the divergence of nuclear BSAP staining of the tumor cells and membrane CD3 staining of reactive T-cells. Double-staining method used: IOP/DAB (brown) (first antibody) and IP)? DAB + NiCl (black) (second antibody). Abbreviations are as in text.

Table 2.

The most common catalytic chromogenic precipitates used for multiple antigen immunostaining

Method	Label	Chromogen	Substrate	Color	References
IGSS	Colloidal gold	(Hydroquinone-silver acetate)		Black	(8–10, 24)
IPO	Peroxidase	3,3′-diaminobenzidine (DAB)	Hydrogen peroxide	Light brown	(5–8, 10, 15, 28–31, 35)
		DAB-nickel chloride		Purple blue	(25, 27)
		DAB-cobal chloride		Dark blue	(7, 25)
		DAB-copper sulfate		Dark brown	(25)
		DAB-nickel chloride-silver nitrate		Grey-black	(26)
		Vector-VIP		Deep violet	(28)
		4-chloro-1-naphthol (4-CN)		Blue	(29, 30)
		3-amino-9-ethylcarbazole (AEC)		Red	(8–10, 15, 36, 37)
IAP	Alkaline phosphatase	Fast red TR	Naphthol-AS-MX-Phosphate	Red	(6, 8, 10, 15)
		Fuchsin Fast blue BB		Magenta	(7, 9, 22, 24)
				Blue	(6–10, 24, 35–37)
		Nitro-blue-tetrazolium (NBT)	5-Bromo-4-chloro-indoxyl-phophate (BCIP)	Purple blue	(15, 36)
IGal	β-d-galactosidase	Ferro-ferri-cyanide	5-Bromo-4-chloro-indoxyl-β-D-galactose	Turquoise	(7, 22, 36)

Preventing Cross-Talk by Inactivation, Hapten-Labeling, Using Monovalent Ig Fab, or Tyramide Amplification

Alternative protocols that combine potentially cross-reacting antibodies focus on either inactivating the immunological layers following the first immunostaining, combining unlabeled and labeled primary antibodies, using monovalent Fab secondary antibodies, or utilizing the sensitivity advantage of tyramide amplification. For inactivating immunological layers of the first sequence, sections may be treated with low pH (pH 1–2) solutions such as 0.01 M hydrochloric acid, a mixture of KMnO₄-H₂SO₄, or oxalic acid (29–31). Other protocols use formaldehyde fixation (32), or microwave heating in an antigen retrieval buffer, between the stains (33, 34). Acidic solutions elute interfering antibodies from the first immunoreaction, however, their effect is not consistent (31). The intermediate application of formaldehyde may either be insufficient, or reduce the antigenicity of the targeted epitopes in the subsequent immunoreactions (34). Although microwave heating between immunostains does not eliminate tissue-bound antibodies, 1–2 × 5 min. of boiling in a 0.01 M citrate buffer (pH 6.0) denatures immunoglobulins to prevent their further reaction in either immunoenzyme or immunofluorescent methods (33, 34). For these reasons, microwave heating between immunostains is the method we favor for minimizing the chance of unwanted color mixing due to potential cross-reaction. Standardized, ready to use, double staining kits with an intermediate blocking step for detecting primary antibodies of the same species are also commercially available, such as the EnVision Double stain kit (Dako, K1395).

Monovalent polyclonal Fab fragments of enzyme coupled secondary immunoglobulins have also been used to reduce or eliminate cross-reactions when using more than one primary antibody from the same species. The polyclonal nature of the secondary allows it to saturate all possible epitopes on the first primary antibody, while the monovalent property of the Fab fragment eliminates the possibility that an unsaturated arm on a divalent secondary may bind subsequent primary antibodies of the same species during their application (35).

The use of directly labeled primary antibodies is also an effective method to eliminate potential cross-reactions (see Chapter 6). Following the detection of the first antibody (e.g., a mouse monoclonal) using a traditional protocol, a FITC- or biotin-labeled second (mouse) monoclonal antibody is applied and detected through its hapten label (i.e., anti-FITC or -biotin) using a different enzyme reaction (24, 36). Here, an intermediate step for saturating anti-mouse Ig binding sites of the first sequence secondary using normal mouse serum is necessary. The commercialization of easy to use mouse Ig biotinylation kits utilizing biotin-coupled monovalent goat anti-mouse-Fab Ig fragments has greatly facilitated this approach (37).

Another method takes advantage of significant differences in detection sensitivities of two immunostaining procedures. For example, the streptavidin-biotin method with biotinylated-tyramide amplification can detect antigens at such a low concentration of the primary antibody, that they can’t be detected with the traditional indirect methods used in the subsequent immunoreactions (38, 39). The principles of double/multiple immunostaining techniques are summarized in Table 3.

Table 3.

Principles of double/multiple immunostaining techniques using simultaneous or sequential immunostaining procedures

Source and type of primary antibody	Blocking of unwanted cross-links	References
Simultaneous procedures (mixing of identical layers)
Noncross-reacting antibodies (e.g., mouse, rabbit, goat etc.)	None	(5, 7, 8, 15)
Same species but different subclass/isotype with isotype-(IgG1, IgG2, IgM etc.)	Detection with isotype-restricted labeled antibodies	(15)
Antibodies of the same species origin	Direct labeling with enzymes or fluorochromes	(4, 16)
	Using preformed complexes with
	Enzyme-labeled anti-immunoglobulins or	(17)
	Alexa-labeled protein A (rabbit Igs)	(18)
Sequential procedures (consecutive immunostainings)
Cross-reacting antibodies (e.g., same species of origin)
Utilizing chromagen shielding effect		(1, 6, 8–10, 24–26, 28)
	IGSS, DAB, DAB-Ni/Co/Cu, DAB-Ni/Ag
Inactivating immune sequences by
	Acidic elution	(29, 30)
	Denaturation with formaldehyde	(32)
	Denaturation by microwave	(33, 34)
Combining unlabeled and hapten-labeled abs
	FITC-labeled, detected with anti-FITC	(24, 36)
	Biotin-labeled, detected with Streptavidin-PO	(36)
	ARK biotinylation with anti-mouse Ig F(ab)	(37)
Using monovalent enzyme labeled F(ab) fragments		(35)
Utilizing sensitivity advantage of tyramide amplification		(38, 39)

The protocols we provide below take advantage of the chromogen shielding effect and the microwave denaturation of immunological sequences to minimize unwanted color mixing, when potentially cross-reacting sequences are used for double or triple immunolabeling.

2. Materials

With the assistance of wet-heat mediated antigen retrieval, most antigens of interest to a general histopathology laboratory can be detected in archival tissues that have been fixed in either 10% buffered formalin (4% formaldehyde) or B5, and embedded in paraffin wax (see Chapter 14). If one or more antigens of interest can not be detected in paraffin embedded tissues, or if the necessary retrieval procedure is incompatible with the detection of another antigen, frozen tissue sections may be used instead (see Chapter 10). Antibodies, immunoreagents, chemicals, and equipment needed are identical to those used for single antigen detection (see Chapters 15–19 and 24–27).

1. Tissue sections of 5 µm thickness. Paraffin sections mounted on commercially available silane-coated glass slides, such as standard SuperFrost Plus (Fischer Scientific, Pittsburgh, PA) or on slides prepared using a home-made APES coating; or frozen sections mounted on silanized, gelatin-coated or poly-L-lysine-coated slides (see Notes 1–4).

2. Lugol’s iodine: 1% iodine and 2% potassium iodine in distilled water.

3. 2.5% and 5% sodium thiosulfate in distilled water.

4. Methanol with 0.5% hydrogen peroxide.

5. Acetone.

6. Antigen retrieval solution (TRS): Target retrieval solution, pH 6.1 (Dako, Carpinteria, CA; catalog number: S1699).

7. Antigen retrieval buffer (citrate): 0.01 M citric acid-sodium citrate, pH 6.0.

8. TBS: 0.05 M Tris–HCl and 0.15 M sodium chloride, pH 7.6.

9. Blocking solution (BS): TBS with 1% bovine serum albumin (BSA), 0.1% sodium azide with 5% normal goat serum.

10. TBS with 0.1% Tween-20; TBS with 0.05% Tween-20.

11. Mouse monoclonal or rabbit polyclonal primary antibodies.

12. Secondary antibodies: goat anti-mouse or anti-rabbit antibodies (1) conjugated with colloidal gold (1.4 nm gold: Nanogold, Stony Brook, NY or 5 nm gold: GE Health Care Life Sciences, Piscataway, NJ; both diluted 1:30 in TBS with 0.05% Tween-20); (2) conjugated with horseradish peroxidase (PO) or alkaline phosphatase (AP) (1:50 dilution in TBS; Dako, Carpinteria, CA); (3) conjugated with separate fluorochromes, e.g., AMCA, FITC, TRITC, Texas Red, Cy-5 (1:50 dilution in TBS; Vector Labs, Burlingame, CA; Jackson ImmunoResearch, West Grove, PA) or with the corresponding Alexa® dyes (1:200 dilution in TBS; Invitrogen, Molecular Probes) (see Tables 1 and 2, Notes 5 and 6).

13. Double-distilled water.

14. Silver acetate developer: ***part a: 250 mg hydroquinone in 50 mL 0.1 M citrate buffer, pH 3.5 (see Note 7); and part b: 100 mg silver acetate (Sigma Aldrich) in 50 mL double distilled H₂O (see Note 8).

15. DAB chromogen-substrate solution: 20 mg DAB (Sigma Aldrich); 100 mL 0.05 M Tris–HCl buffer, pH 7.6; and 100 µL 30% H₂O₂. Alternatively, standardized DAB+ chromogen-substrate kit (e.g., Dako, code: K3467) (see Subheading 3.3.2).

16. Nickel chloride salt (Sigma Aldrich).

17. AEC chromogen-substrate solution: AEC (Sigma Aldrich); N,N-dimethylformamide; 0.1 M acetate buffer pH 4.6; and hydrogen peroxide. Alternatively, standardized AEC+ chromogen-substrate kit (e.g., Dako, code: K3461) (see Subheading 3.3.4).

18. Fast Blue chromogen-substrate solution: Fast Blue BB Salt (Sigma); Naphthol AS-MX-phosphate (Sigma); N,N-dimethylformamide; 0.1 M Tris–HCl buffer, pH 9.0; and 1 M Levamisole (see Note 9) (see Subheading 3.3.5).

19. BCIP/NBT standardized chromogen-substrate kit (e.g., Dako, code: K0598).

20. Fast Red-naphthol phosphate standardized chromogen-substrate kit (e.g., Dako, code: K0597).

21. Fuchsin-naphthol phosphate standardized chromogen-substrate kit (e.g., Dako, code: K0624).

22. Glycerol-based mounting medium containing antifading agent, e.g., Vectashield (Vector Labs.); or glycerol-gelatin based mounting medium, e.g., Faramount (Dako) (see Note 10).

3. Methods

3.1. Sequential Antigen Detection

In sequential detection protocols (see Note 11), the immunohistochemical detection of each antigen is performed separately and sequentially. As discussed above in Subheading 1.1.3.1, the precipitated reaction products of IGSS, IPO or IAP can mask the immunological sequences of their associated antigen-antibody complex, thereby preventing secondary antibodies used in the subsequent detections from binding to primary antibodies of identical species. Microwaving between the stains in citrate buffer (pH 6.0) can optionally be used to inactivate previously applied reagents. Primary antibodies of the same animal species and/or cross-reacting antibodies can be combined, provided that the antigens are localized in different cell types or in different areas of the same cell. Immunofluorescence methods should not be used in combinations with cross-reacting antibodies.

For paraffin sections begin with step 1 and skip steps 5 and 6; for frozen sections begin with step 5 (see Notes 1–4 and 12) (see Chapters 10 and 13).

1. Dewax paraffin sections in three changes of xylene and ethanol for 2 min each.

2. Apply wet-heat mediated antigen retrieval in TRS, pressure cooking or microwaving when appropriate (see Chapter 14).

3. Place sections in Lugol’s iodine for 5 min, then in 2.5% sodium thiosulfate for 30 s if IGSS method is to be used.

4. Block endogenous peroxidase in methanol containing 0.5% hydrogen peroxide for 15 min.

5. Allow freshly cut frozen sections to dry for ~15 min at room temperature.

6. Fix in acetone for 5–10 min and let sections dry again for at least 2 h at room temperature before use. Drying can be shortened to 3–5 min by using a hair dryer with constant agitation from a 5–10 cm distance.

7. Wash dewaxed or frozen sections in TBS for 3 min and incubate in blocking solution (BS) for 5–10 min. Use BS for diluting primary antibodies and TBS for diluting all other immunoreagents.

8. Apply primary antibody (mouse monoclonal or rabbit polyclonal) for 2 h on paraffin sections and for 30 min on frozen sections at room temperature (see Note 13).

9. Wash slides for three times for 2 min each in TBS containing 0.1% Tween-20. Acetone-fixed frozen sections must be washed with neat TBS.

10. Apply goat anti-mouse or anti-rabbit secondary antibody labeled either with colloidal gold or horseradish peroxidase for 30 min (see Notes 5 and 14).

11. Wash for two times for 2 min each in TBS containing 0.1% Tween 20. Acetone-fixed frozen sections must be washed with neat TBS.

12. Before silver amplification of gold particles rinse the sections three times for 30 s and then wash three times for 2 min each in double-distilled water.

13. Use silver development for IGSS (see Notes 15 and 16) or DAB chromogenic-substrate reaction for IPO as detailed in Subheadings 3.3.1 and 3.3.2.

14. Optional for paraffin sections if cross-reacting antibody follows: Rinse slides in TBS and apply an intermediate microwave heating at 300 W power in 150 mL of citrate buffer (pH 6) for 10 min. Cool in flowing tap water.

15. Incubate sections in BS for 5–10 min then apply the primary antibody of the second immunological sequence for 2 h on paraffin sections or for 30 min on frozen sections.

16. Wash three times for 2 min each with TBS and apply the appropriate secondary antibody; that is goat anti-rabbit or anti-mouse Ig coupled either to peroxidase or alkaline phosphatase, for 30 min (see Note 17).

17. Wash slides three times for 2 min each in TBS.

18. Develop the chromogenic reaction of the second sequence using either AEC chromogenic substrate reaction for IPO or naphthol phosphate/Fast Blue, NBT/BCIP, Fast Red or New Fuchsin for IAP development as detailed in Subheading 3.3 (see Note 18 for triple labeling).

19. Mount sections, without dehydrating when any IPO/AEC or any IAP detection method was involved, using Faramount (Dako).

3.2. Simultaneous Antigen Detection

In this method, the primary antibodies are combined into a single cocktail and applied simultaneously in a single-reaction step. The same can be done for labeled secondary antibodies. Antibodies of different animal species, or different immunoglobulin isotypes (noncross-reacting) detected with isotype-restricted labeled immunoglobulins, or antibodies of the same species directly coupled to different enzymes/fluorochromes are required. Immunofluorescent methods are particularly suited for this method.

Steps 1–7 are identical to those described in Subheading 3.1.

1. Mix primary antibodies (e.g., a rabbit polyclonal with a mouse monoclonal) in BS. Prepare double concentrations of half the needed volume from each primary antibody, mix equal volumes, and apply the primary antibody mixture onto the sections. Incubate paraffin sections for at least 2 h and frozen sections for 30 min.

2. Wash slides for three times for 2 min each in TBS containing 0.05% Tween-20. Acetone-fixed frozen sections must be washed in neat TBS.

3. Apply appropriate, labeled secondary antibodies together, (e.g., goat anti-mouse and anti-rabbit immunoglobulins), labeled either with colloidal gold, horseradish peroxidase or alkaline phosphatase, respectively. Alternatively, use secondary antibodies labeled with separate fluorochromes (see Table 1). Incubate paraffin sections for 1–2 h and frozen sections for 30 min or less.

4. Wash for three times for 2 min each in TBS.

5. Use the relevant visualization steps described in Subheading 3.3 to develop chromogenic precipitates for IGSS, IAP, or IPO techniques, sequentially.

6. Cell nuclei may be counterstained either with DAPI (blue) or 7-AAD (red) after immunofluorescence stainings (see Note 19), or with hematoxylin or methyl green after immunoenzyme techniques.

7. Rinse slides and mount them with glycerol-gelatin based Faramount (Dako) without dehydration.

3.3. Visualization of Reaction Products for Light Microscopy

The wide range of chromogenic-substrate systems available allows one to obtain excellent color contrast for double/multiple antigen detection (see Tables 1 and 2). (see Note 20).

3.3.1. IGSS

1. Prepare silver acetate developer (40) solution a by dissolving 250 mg hydroquinone in 50 mL 0.1 M citrate buffer, pH 3.5; and solution b by dissolving 100 mg silver acetate in 50 mL double-distilled water (see Notes 7 and 8).

2. Mix a and b above just before use.

3. Immerse sections in developer and place the jar in a dark place. After 5–8 min monitor the intensity of the gray-black product under the microscope.

4. When desired intensity is achieved, wash slides in distilled water for 2 min, and then in 5% sodium thiosulfate for 2 min.

3.3.2. IPO/DAB

1. Dissolve 20 mg DAB in 100 mL 0.05 M Tris–HCl buffer, pH 7.6, and mix 100 µL 30% hydrogen peroxide in it just before use.

2. Place slides into DAB solution in Coplin jar and develop for 5–15 min under microscopic control to obtain a yellow-brown product.

Alternatively, use DAB+ chromogen-substrate kit available from Dako (or other comparable kit) by mixing 20 µL of DAB chromogen per 1 mL of substrate buffer. Pipette 100 µL mixture on the horizontally placed sections and develop for 5–15 min.

3.3.3. IPO/DAB-Ni

1. Dissolve 20 mg DAB and 0.5% nickel chloride in 100 mL 0.05 M Tris–HCl buffer, pH 7.6, and mix 100 µL hydrogen peroxide in it just before use.

2. Develop slides for 5–15 min under microscopic control to obtain a purple-black product (see Note 21).

3.3.4. IPO /AEC

1. Mix 2.5 mL of AEC dissolved in dimethylformamide into 90 mL of 0.1 M acetate buffer, pH 4.6. Mix 100 µL 30% hydrogen peroxide with the AEC solution before use.

2. Place slides into AEC solution in a Coplin jar and develop for 5–15 min under microscopic control to obtain a red-brown product. Alternatively, drop AEC+ “ready-to-use” chromogen/substrate solution available from Dako (or other comparable kit) onto the sections and develop for 5–15 min.

3.3.5. IAP/Fast Blue

1. Dissolve 2 mg naphthol AS-MX phosphate (Sigma) in 200 µL N,N-dimethylformamide, mix solution with 9.8 mL 0.1 M Tris–HCl buffer (pH 9.0) and add one drop of 1 M levamisole (see Note 9).

2. Before use, add 10 mg of Fast Blue BB salt, shake to dissolve, filter, and drop the mixture onto the sections. A blue product will form in 5–15 min.

3.3.6. IAP/NBT-BCIP (See Note 22)

1. Apply “ready-to-use” reagent mix available from Dako (or other comparable kit) onto the section, and develop between 10 min and several hours to produce a purple blue reaction product on the specific antigenic sites (see Note 23).

3.3.7. IAP/Fast Red (See Note 22)

1. Using a commercial kit available from Dako (or other comparable kit) dissolve one tablet containing the Fast Red chromogen and levamisole in 3 mL of 0.1 M Tris–HCl, pH 8.2 substrate buffer provided in the kit.

2. Drop the mixture onto the sections and develop for 5–30 min to get a magenta-red reaction product.

3.3.8. IAP/Fuchsin (See Note 22)

1. Using a commercial kit available from Dako (or other comparable kit) add one drop Tris-buffer concentrate (vial a) and one drop of substrate concentrate (vial b) to 2 mL of distilled water.

2. Mix one drop Fuchsin (vial c) with one drop sodium nitrite (vial d). After 2 min, mix the chromogen solution with the substrate-buffer. Cover tissue sections with the solution. A magenta-red product will form in 5–15 min.