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Application of Antigen Retrieval by Heating for Double-label Fluorescent Immunohistochemistry with Identical Species-derived Primary Antibodies

Hidetoshi Ino

Department of Neurobiology (C1), Graduate School of Medicine, Chiba University

Correspondence to: Hidetoshi Ino, Dept. of Neurobiology (C1), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: h.ino{at}faculty.chiba-u.jp

	Summary

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Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies. (J Histochem Cytochem 52:1209–1217, 2004)

Key Words: immunofluorescence • multiple labeling • double labeling • monovalent antibodies

	Introduction

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DEMONSTRATION of co-localization of independent antigens in identical cells or cell compartments is one of the essential techniques in cell biology. For this, usually double and occasionally triple immunohistochemical (IHC) and immunocytochemical (ICC) labeling are performed. Antibodies derived from different animal species are easily distinguished by secondary antibodies against species-specific immunoglobulins. In general, double-labeling techniques are based on this principle, and sets of primary antibodies derived from different animal species are used. However, when pairs of primary antibodies are antibodies derived from the same species, the general double-labeling methods cannot be used.

To enable double labeling with primary antibodies from the same species, several methods have been developed: (a) adjacent thin sections are separately stained and compared (serial sectioning and mirror sectioning methods; Osamura et al. 1981; Tsutsumi 1988; Stevenson and Pflüger 1994); (b) primary antibodies are monoclonal antibodies belonging to different immunoglobulin subclasses (Tidman et al. 1981); (c) primary antibodies are directly labeled with enzymes, fluorophores, or haptens such as biotin (direct labeling method; Valnes and Brandtzaeg 1982; Boorsma 1984; Van der Loos et al. 1989; Würden and Homberg 1993); (d) primary and secondary antibodies are sequentially applied and stained (sequential staining method; Falini et al. 1986; Wessel and McClay 1986; Franzusoff et al. 1991; Lewis Carl et al. 1993; Negoescu et al. 1994; Hontanilla et al. 1997); (e) highly sensitive tyramide signal amplification is used to distinguish primary antibodies (Shindler and Roth 1996; Hunyady et al. 1996; Brouns et al. 2002; Uchihara et al. 2003). Method a is suitable only for large cells spanning to two sections, which limits the extent of application. Method b requires highly specific secondary antibodies to immunoglobulin subclasses and is inapplicable to polyclonal antibodies or to monoclonal antibodies belonging to the same subclass. Although method c is applicable in any case, the preparation of labeled primary antibodies is laborious. Method d is superior to the former methods in the extent of application and in the easiness of procedure, but it is not so easy to exclude false-positive staining by secondary antibodies' crossreaction or insufficient removal of first primary antibodies. To avoid secondary antibodies' crossreaction, methods using monovalent secondary antibodies, especially using Fab fragments, have been reported (Wessel and McClay 1986; Franzusoff et al. 1991; Lewis Carl et al. 1993; Negoescu et al. 1994), and kits for these methods are supplied from some manufacturers, such as Zenon labeling reagents from Molecular Probes (Eugene, OR). Method e works when a first primary antibody is applied at a low concentration, which is detectable by tyramide amplification but not with a fluorescently labeled secondary antibody. A second primary antibody is applied at a normal concentration. Because the second primary antibody is applied after tyramide amplification of the first primary antibody, these two antibodies can be distinguished. Although this system is excellent, it is applicable to double but not to triple labeling.

On the other hand, immunostaining methods using soluble immune complexes consisting of primary and secondary antibodies have been reported (Tuson et al. 1990; Krenács et al. 1991; Eichmüller et al. 1996). By combining the method using monovalent secondary antibodies and that using soluble immune complexes, an improved method for double-label IHC with identical species-derived primary antibodies is obtained. With this method, secondary antibodies' crossreaction is avoidable and the required amount of secondary antibodies can be saved. There are two procedures for this double-labeling method. The reaction is completed by two-step incubation as follows (Figure 1A) : a first primary antibody (M) is applied to sections, followed by the reaction with a first monovalent secondary antibody (X). A soluble immune complex consisting of a second primary antibody (N) and a second monovalent secondary antibody (Y) is formed before application to sections. After absorption of the excess second secondary antibody (Y) with nonspecific immunoglobulin, the immune complex is applied to sections. Alternatively, the reaction is completed by one-step incubation (Figure 1B): two soluble immune complexes consisting of a primary antibody and a monovalent secondary antibody are separately formed, and the excess secondary antibodies are absorbed. Then the immune complexes are applied to sections.

View larger version (14K): [in this window] [in a new window]   Figure 1 Schematic representation of double-label fluorescent IHC with monovalent secondary antibodies. (A) Two-step incubation procedure. A first primary antibody (M) is reacted with an antigen (m) in sections and then a first monovalent secondary labeled with X is applied. After the first reaction, an immune complex consisting of a second first antibody (N) and a second monovalent secondary antibody labeled with Y is applied. (B) One-step incubation procedure. Two immune complexes are separately formed and then applied to sections. In each procedure, the excess secondary antibodies in the immune complexes are absorbed with nonspecific immunoglobulin before application to the sections.

	Materials and Methods

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Preparation of Tissue Sections
Male Sprague-Dawley rats (body weight 250–300 g) were perfused via the heart with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.5, under deep pentobarbital anesthesia. All animals were treated and cared for in accordance with the Chiba University School of Medicine guidelines pertaining to the treatment of experimental animals. Brains and dorsal root ganglia (DRG) were collected. Forebrains and brainstems were coronally sliced at 5-mm thickness and cerebella were sagittally cut. The tissues were postfixed in the same fixation solution at 4C for 1 day, incubated in PBS at 4C for 1 day, and incubated in 30% sucrose in PBS at 4C for 1 day. The tissues were embedded in OCT Compound (Sakura Fine Technical; Tokyo, Japan) and frozen with dry ice. Frozen sections were cut with a cryostat at 10-µm thickness and mounted on glass slides coated with poly-L-lysine. After drying in a vacuum desiccator for more than 1 day, the sections were used for IHC. For some tissues, AR by heating en bloc was performed after postfixation, as follows.

AR by Heating
AR by heating en bloc was performed for some tissues as previously described (Ino 2003,2004). After postfixation, tissues were incubated in distilled water at 4C for 1 day and boiled in distilled water for 3 min. The tissues were then placed in ice-cold 30% sucrose in PBS and incubated in the same solution at 4C for 1 day. Frozen sections were prepared as above.

Primary Antibodies
Primary antibodies are listed in Table 1. In total, 12 antibodies were tested, among which five monoclonal antibodies were raised in mouse (IgG₁) and seven polyclonal antibodies were raised in rabbit or guinea pig.

Preparation of Soluble Immune Complexes
A primary antibody and a 5-µg/ml divalent or monovalent secondary antibody were reacted in 1.5% Blocking Reagent (Roche) in PBS (buffer A) at RT for 2 hr to form a soluble immune complex. Secondary antibodies were labeled with either biotin or DIG.

Immunohistochemical Procedures
Sections were incubated in 0.3% Triton X-100 in PBS at RT for 3 hr, and blocking was performed in 5% skim milk in PBS at RT for 4–6 hr. The sections were incubated with biotin-labeled soluble immune complexes at RT overnight. After washing with PBS, the sections were incubated with AlexaFluor 488-conjugated streptavidin (1:400; Molecular Probes) in buffer A at RT for 2 hr. After washing with PBS, the sections were mounted with the mounting medium Parma Fluor (Thermo Shandon; Pittsburgh, PA) and observed with a fluorescence microscope. Alternatively, IHC was performed by the general sequential procedure as control experiments. After blocking with 5% skim milk in PBS, sections were incubated with a primary antibody in buffer A at RT for 3 hr to overnight. After washing in PBS, the sections were incubated with a 5-µg/ml biotin-conjugated divalent or monovalent secondary antibody in buffer A at RT for 3 hr. The following procedure was as above.

Levels of immunoreactivity were scored as negative (–), equivocal (±), weak (+), moderate (++), and strong (+++).

Double-label Fluorescent IHC (Two-step Incubation)
The procedures of double-label fluorescent IHC are summarized in Figure 2 . After blocking with 5% skim milk in PBS, sections were incubated with a first primary antibody in buffer A at RT for 3 hr to overnight. After washing with PBS, the sections were incubated with a 5-µg/ml first DIG-conjugated monovalent secondary antibody in buffer A for 3 hr. After forming a biotin-labeled soluble immune complex with second primary and secondary antibodies, nonspecific mouse immunoglobulin (final concentration 1 mg/ml; Sigma, St Louis, MO) was added to the immune complex to absorb the excess secondary antibody and the solution was incubated at RT for 1 hr. After washing with PBS, the sections were incubated with the immune complex solution at RT for 3 hr. After washing with PBS, the sections were further incubated with AlexaFluor 488-conjugated streptavidin (1:400) and rhodamine-conjugated anti-DIG antibody (1:200; Roche) in buffer A at RT for 2–3 hr. The sections were mounted and observed as above.

View larger version (31K): [in this window] [in a new window]   Figure 2 Brief protocol for double-label IHC with identical species-derived primary antibodies. Two-step and one-step incubation procedures are summarized in flow charts. Antibodies were incubated in 1.5% Blocking Reagent in PBS. The concentration of secondary antibodies was 5 µg/ml. The concentrations or dilutions of primary antibodies were as described in Table 1. The incubation was performed at RT for 3 hr to overnight. aPreparation of AR(+) sections was as previously described (Ino 2003). bDIG and biotin labeling of secondary antibodies are interchangeable in two-step incubation.

	Results

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IHC with Preformed Soluble Immune Complexes
One of the important points in the present method is whether preformed soluble immune complexes consisting of primary and secondary antibodies react to antigens in sections. To address this question, I examined the immunoreactivity of soluble immune complexes using a number of primary antibodies listed in Table 1, including mouse monoclonal (IgG₁ isotype) and rabbit and guinea pig polyclonal antibodies. Secondary antibodies were composed of divalent anti-mouse, -rabbit, and -guinea pig IgG(H+L) antibodies, monovalent anti-mouse IgG(H+L) Fab fragments, and monovalent anti-mouse, -rabbit, and -guinea pig IgG(Fc) Fab fragments (Table 2). The anti-IgG(Fc) antibodies specifically recognize the Fc region of an antibody, whereas the anti-IgG(H+L) antibodies recognize the entire IgG molecule, including the heavy and light chains. All the secondary antibodies used in this experiment were labeled with biotin. Sections were from the rat forebrain, brainstem, cerebellum, and DRG tissues. Some of the tissues received AR by heating en bloc before preparation of cryostat sections [AR(+)] and the others did not [AR(–)].

Before examining the immunoreactivity of the soluble immune complexes and the effects of AR, I confirmed that all the primary antibodies used here showed strong immunoreactivity by the general sequential labeling procedure on AR(+) sections, although weaker immunoreactivity was observed for NeuN, GFAP (monoclonal and polyclonal), TH, and c-Fos on AR(–) sections (data not shown). Equivalent levels of immunoreactivity were observed for the other primary antibodies either on AR(+) or AR(–)sections (data not shown). The conditions for AR were fixed in the following experiments (heating at boiling temperature for 3 min in distilled water), because the effects of AR for IHC by the general sequential labeling procedure were remarkable under these conditions.

The results are shown in Table 3. The anti-CBP and anti-parvalbumin monoclonal antibodies showed moderate immunoreactivity on AR(–) sections in the form of soluble immune complex regardless of the sort of the secondary antibodies (CBP was located in Purkinje cells and parvalbumin was located in Purkinje cells, stellate cells, and basket cells in the cerebellum). AR enhanced the immunoreactivity up to equivalent levels demonstrated by the general sequential labeling procedure. On the other hand, as to the anti-NeuN, GFAP, and TH monoclonal antibodies, no immunoreactivity was observed with the soluble immune complexes including the divalent secondary antibody on either AR(–) or AR(+) sections. Even with the immune complex including the anti-IgG(H+L) Fab or anti-IgG(Fc) Fab, occasionally only weak (NeuN and TH) or no (GFAP) staining was detected on AR(–) sections. However, AR enhanced the immunoreactivity to moderate levels, although the immunoreactivity was lower than that seen with the general sequential labeling procedure (NeuN and GFAP immunoreactivity was observed in neurons and astrocytes, respectively; TH immunoreactivity was seen in dopaminergic neurons). As to the anti-GFAP polyclonal antibody, although weak immunoreactivity was observed on AR(+) sections with the soluble immune complex including the divalent secondary antibody, the enhancement of the immunoreactivity by AR was more apparent with use of the soluble immune complex including the monovalent secondary antibody. These results indicate that AR was critical for successful immunostaining for NeuN, GFAP (monoclonal and polyclonal), and TH in the form of a soluble immune complex. As to the secondary antibodies, no difference was observed between the anti-IgG(H+L) Fab and anti-IgG(Fc) Fab. In contrast to the above primary antibodies, no immunoreactivity was observed with the immune complexes for the other primary antibodies (c-Fos, Egr-1, CGRP, substance P, VR1, and P2X3) either on AR(–) or AR(+) sections, although these antibodies showed moderate to strong immunoreactivity by the general sequential labeling procedure regardless of AR (data not shown; c-Fos and Egr-1 immunoreactivity was observed in nuclei of several neurons; CGRP, substance P, VR1, and P2X3 were located in neurons of the DRG).

In any case, no immunoreactivity was observed in the absence of the primary antibodies (data not shown).

Double-label Fluorescent IHC
I show here two examples of double-label fluorescence IHC. First, the immunoreaction was performed by two-step incubation using the anti-NeuN monoclonal antibody as a first primary antibody, the anti-GFAP monoclonal antibody as a second primary antibody, the DIG-conjugated anti-mouse IgG(Fc) Fab fragment as a first secondary antibody, and the biotin-conjugated anti-mouse IgG(Fc) Fab fragment as a second secondary antibody. To absorb the excess second secondary antibody in the immune complex, 200-fold amounts of nonspecific mouse immunoglobulin were added after the formation of the immune complex. NeuN immunoreactivity was observed in neurons and GFAP immunoreactivity was seen in astrocytes, and no crossreactivity was found (Figures 3A–3C) . Next, the immunoreaction was performed by one-step incubation using the anti-CBP and parvalbumin monoclonal antibodies as primary antibodies and the DIG- and biotin-conjugated anti-mouse IgG(Fc) Fab fragments as secondary antibodies. The absorption of the excess secondary antibodies was performed as above. As described, CBP is located in Purkinje cells, and parvalbumin is located in Purkinje cells, stellate cells, and basket cells in the cerebellum. Figures 3D–3F show CBP immunoreactivity in Purkinje cells and parvalbumin immunoreactivity in Purkinje cells, stellate cells, and basket cells. Crossreaction with the anti-CBP antibody was not observed in stellate and basket cells (Figure 3F, arrowheads).

View larger version (89K): [in this window] [in a new window]   Figure 3 Double-label fluorescent IHC with primary antibodies derived from identical species. (A–C) NeuN (A) and GFAP (B) were stained on an AR(+)section of the rat amygdala by two-step incubation. The two images are merged in C. The mouse anti-NeuN monoclonal antibody was used as the first primary antibody, and the mouse anti-GFAP monoclonal antibody was as the second primary antibody. The DIG-conjugated anti-mouse IgG(Fc) Fab fragment was used as the first secondary antibody, and the biotin-conjugated anti-mouse IgG(Fc) Fab fragment was as the second secondary antibody. The anti-NeuN antibody and the DIG-conjugated anti-mouse IgG(Fc) Fab fragment were sequentially applied to the section. After the reaction of the first antibodies, the immune complex consisting of the anti-GFAP antibody and biotin-labeled anti-mouse IgG(Fc) Fab fragment was applied to the section. No overlapping staining was observed between NeuN and GFAP (C). (D–F) CBP (D) and parvalbumin (E) were stained on an AR(+) section of the rat cerebellum by one-step incubation. The two images are merged in F. The first immune complex consisted of the mouse anti-CDP monoclonal antibody and DIG-conjugated anti-mouse IgG(Fc) Fab fragment, and the second of the anti-parvalbumin monoclonal antibody and biotin-conjugated anti-mouse IgG(Fc) Fab fragment. The immune complexes were separately formed and then simultaneously applied to the section. Purkinje cells (F, arrows) were positive for both CBP and parvalbumin, while stellate cells and basket cells (F, arrowheads) were positive only for parvalbumin. Signal was detected with the rhodamine-conjugated anti-DIG Fab fragment and AlexaFluor 488-conjugated streptavidin. Bar = 50 µm.

	Discussion

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The reduced or abolished immunoreactivity of the soluble immune complexes is likely to be due to the increased size of the immune complexes. In the second group of the antibodies, the soluble immune complexes with the divalent secondary antibody showed no or weak immunoreactivity even in AR(+) sections, whereas those with the monovalent Fab secondary antibodies showed moderate immunoreactivity in AR(+) sections. Because of the increased molecular size, the immune complexes may be hindered from approaching to antigens fixed in sections, and that may be the reason why AR by heating, which breaks the higher structure around the antigens, is effective for immunostaining. Because the Fc fragment is located more distantly from the recognition sites of the antibody than the Fa and Fb fragments, I had expected that secondary antibodies attached to the Fc fragment of primary antibodies would show less interference in antibody–antigen interaction of primary antibodies and that the Fab anti-IgG(Fc) antibody would show stronger immunoreactivity than the Fab anti-IgG(H+L) antibody. However, in practice there was no difference in immunoreactivity between these two monovalent secondary antibodies, at least within the primary antibodies I examined. There is a possibility that the ratio of primary antibody to secondary antibody is critical for the immunoreactivity of soluble immune complexes (Van der Loos and Göbel 2000). However, calculation of this ratio is usually difficult because the concentration of commercially supplied primary antibodies is mostly unknown.

It is impossible to predict which primary antibody shows positive immunoreactivity in the form of a soluble immune complex. Both cases, positive and negative, were observed in the antibodies against cytoplasmic and nuclear proteins. It was surprising that antibodies against small peptides, such as CGRP and substance P, showed no immunoreactivity in the form of a soluble immune complex.

As described, without AR this double-labeling method can be used only in restricted cases. However, AR expands the selective range of primary antibodies for this method. If one of two antibodies can react in AR(+) sections in the form of a soluble immune complex, double labeling can be performed by two-step incubation using the positive antibody as a second primary antibody.

	Acknowledgments

 
I thank Ms Yoko Hata for valuable technical support.

	Footnotes

 
Received for publication November 10, 2003; accepted April 7, 2004

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