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Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections

Shuji Yamashita and Yasunori Okada

Electron Microscope Laboratory (SY) and Department of Pathology (YO), School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan

Correspondence to: Shuji Yamashita Electron Microscope Laboratory, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo 160-8582. E-mail: shuji{at}sc.itc.keio.ac.jp

	Summary

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We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl₂ for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl₂, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor (ER) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ER immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl₂ at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections. (J Histochem Cytochem 53:1421–1432, 2005)

Key Words: heat-induced antigen retrieval • fresh frozen section • unoccupied steroid hormone • receptors • immunohistochemistry

	Introduction

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HEAT TREATMENT IS THE MOST COMMON method for the antigen retrieval in immunohistochemical studies of formalin-fixed and paraffin-embedded tissues (Shi et al. 1991,2001; Cattoretti et al. 1993; Werner et al. 1996; Pileri et al. 1997). Heat-induced antigen retrieval (HIAR) is also applied for prefixed and unfrozen specimens (Yamashita et al. 1989; Evers and Uylings 1997; Evers et al. 1998; Ino 2003). Recent studies using SDS-PAGE by Rait et al. (2004) and us (2005) have demonstrated that heat treatment cleaves the formalin-induced intra- and intermolecular crosslinks of proteins. We have also shown by a Western blot technique that immunoreactive ß-actin and fibronectin can be extracted from autoclaved paraffin sections in an SDS and urea solution, but not from unheated sections. These results indicate that the main mechanism of HIAR is based on the disruption of formaldehyde-induced crosslinks (i.e., methylene bridges). Epitopes located on the inner portion of antigen molecules or hidden by other oligomeric components may also be exposed by heat denaturation, even though tissues are not treated with chemical fixatives (Yasuda et al. 1986; Yamashita et al. 1989,1997). Furthermore, antibodies may easily penetrate into the tissues to interact with antigens, because a gellike structure formed by the crosslinks is cleaved by heating.

Fresh frozen sections are widely used for immunohistochemical studies, because (1) they preserve antigenecitiy well, (2) they are convenient for comparing antigen expression in the different size of tissues with a constant fixation time, and (3) they allow antigen localization in a short time for pathological diagnosis. In particular, frozen sections fixed with formalin or acetone have been used as a standard to evaluate the efficiency of HIAR in formalin-fixed and paraffin-embedded specimens (Shi et al. 1993; Merz et al. 1995; Mighell et al. 1995), whereas HIAR has not applied to the frozen sections, probably because they are fragile and readily destroyed by heating (Ino 2003). However, if HIAR is based on these mechanisms, it should also be useful to immunohistochemical studies in frozen sections mounted on slide glasses. The main differences between frozen sections and paraffin sections may be as follows: protein denaturation in frozen sections is less intense than in paraffin sections, because proteins are not exposed to organic solvents and heat, and macromolecules may be more extracted from frozen sections during heat-treatment compared with those in paraffin sections.

Fixation is one of the most important factors in immunohistochemistry, and both excess fixation and insufficient fixation weaken immunostaining. Soluble antigens such as ligand-free steroid hormone receptors are readily extracted from fresh frozen sections during fixation. In the present study, we tried to establish a procedure for rapid and complete immobilization of antigens in fresh frozen sections and successive antigen retrieval by heating, and to investigate the mechanisms of HIAR in detail. The procedure was also applied to localize unliganded estrogen receptor (ER) and glucocorticoid receptor (GR), which are known to be readily extracted from fresh frozen sections during fixation (Gasc et al. 1989; Yamashita and Korach 1989; Pekki et al. 1992).

	Materials and Methods

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Reagents
The primary antibodies used in this study, their respective sources and dilutions are listed in Table 1. Envision plus/horseradish peroxidase for rabbit immunoglobulin and mouse immunoglobulin were purchased from DakoCytomation (Carpinteria, CA), and anti-goat IgG rabbit antibody was from ICN Biomedicals, Inc (Aurora, OH). A blocking reagent (block ace) was obtained from Dainippon Pharmaceutical Co., Ltd (Osaka, Japan). Formalin and 10% glutaraldehyde were purchased from Wako Pure Chemicals Industries Ltd (Osaka, Japan).

To detect liganded and unliganded ER, immature female mice (2 weeks old) were intraperitoneally injected with a single dose of 0.1 ml 17ß-estradiol (E2, 20 µg/kg BW) or vehicle solution (1% ethanol and 99% saline) and were killed 1 hr after the injection. To localize hormone-free and hormone-bound GR, female mice (10 weeks old) were adrenalectomized, and 7 days later they were intraperitoneally given 0.2 ml of 70% ethanol containing 25 µg of dexamethasone or vehicle solution, and sacrificed after 1 hr. Four hormone-injected mice and four vehicle-injected control mice were used for each experiment.

Immunohistochemistry
Fixation Protocols and Antigen Retrieval
Fresh frozen sections (6 µm thick) mounted on new silane-coated slide glasses (Muto Pure Chemicals Co., Ltd.; Tokyo) were fixed with the following fixatives for 1–360 min at room temperature or 40C: (1) 10% or 20% formalin dissolved in 0.1 M cacodylate buffer (CB) or 0.1 M phosphate buffer (PB) (pH 7.4); (2) 10% or 20% formalin in 0.1 M CB (pH 7.4) containing 5–100 mM CaCl₂; (3) 0.5%, 1%, or 2% glutaraldehyde in 0.1 M PB (pH 7.4); and (4) ice-cold acetone. The sections fixed with aldehyde were washed with TBS (10 mM Tris-HCl buffer, pH 7.4, containing 0.85% NaCl). For HIAR, the sections were boiled in a microwave oven for 20 min or autoclaved for 10 min in 20 mM Tris-HCl buffer (TB), pH 9.0: our previous study showed that most antigens yielded the strongest immunoreaction when sections were heated at pH 9.0 (Yamashita and Okada 2005; Emoto et al. 2005). Allowing them to cool to room temperature, the sections were washed with PBS. To detect -amylase, lysozyme, insulin-like growth factor binding protein-3, and common antigen of secretory granule membrane, the aldehyde-fixed fresh frozen sections were treated with 0.05% Triton X-100 in PBS for 5 min for membrane permeabilization. The acetone-fixed sections were air-dried and then immersed in PBS. The sections were then incubated with a blocking solution (i.e., 1% BSA and 10% block ace in PBS) for 60 min successively, with the primary antibodies diluted with the blocking solution overnight at 4C. When the primary antibodies had been raised in goats, the sections were further treated with anti-goat IgG rabbit antibody (1:500) for 30 min. The sections were then incubated with Envision plus for rabbit or for mouse for 60 min at room temperature. Peroxidase enzyme activity was detected with imidazole-3, 3'-diaminobenzidine solution (Yamashita and Korach 1989).

Normal rabbit IgG or mouse IgG was used in place of the primary antibody for control immunostaining. For the control of ER, ERß, GR, and androgen receptor (AnR) immunostaining, antibodies absorbed with the respective antigenic peptides were used.

Effect of EDTA on Sections Fixed with Formalin Containing CaCl₂
To investigate the effect of calcium ions on fixation, the sections fixed with formalin containing 25 mM CaCl₂ for 30 min at room temperature, were treated with or without 1 mM EDTA in 20 mM TB (pH 9.0) at room temperature for 2 hr, or autoclaved in the buffer in the presence or absence of 1 mM EDTA for 10 min, and then immunostained with the antibodies.

	Results

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Morphological Effects of Fixatives and Heating
The sections fixed with acetone and immunostained with normal rabbit IgG as a control maintained relatively good morphology, although nuclear staining with hematoxylin was weaker than that of formalin-fixed fresh sections (Figures 1A and 1B). When fresh frozen sections were fixed with 10% formalin in 0.1 M PB or CB for 30–360 min at room temperature or at 40C and then boiled in a microwave oven or autoclaved, the nuclear structure of many cell types, particularly the epithelial cells of the genital tract and intestine, was destroyed, indicating that chromatin had been extracted (Figures 1B and 1C). However, the nuclear structure in the sections fixed with 10% formalin containing CaCl₂ was well preserved after heating (Figure 1D), and fixation with 10% formalin containing 25 mM CaCl₂ for 30 min yielded much better morphology than fixation with 10% formalin for 5 hr (Figures 1C and 1D). Glutaraldehyde was an excellent fixative for preservation of morphology. Fixation with 0.5% glutaraldehyde even for 1 min at room temperature prevented chromatin extraction after autoclaving (Figure 1E).

View larger version (171K): [in this window] [in a new window]   Figure 1 Morphological effect of fixatives and heating. Fresh frozen sections from mouse epididymis were fixed with ice-cold acetone for 30 min, successively dried, and then immunostained with normal rabbit IgG as a control (A). Frozen sections were fixed with 10% formalin in 0.1 M phosphate buffer (PB), pH 7.4, for 30 min (B) or 5 hr (C), with 10% formalin in 0.1 M cacodylate buffer (CB), pH 7.4, containing 25 mM CaCl2 for 30 min (D), or with 0.5% glutaraldehyde in 0.1 M PB, pH 7.4, for 1 min at room temperature (E). The sections were stained with hematoxylin and eosin after autoclaving at 120C for 10 min in 20 mM Tris HCl buffer, pH 9.0, (C–E) or without autoclaving (A,B). Bar = 50 µm.

View larger version (126K): [in this window] [in a new window]   Figure 2 p300 and claudin-1 immunostaining in fresh frozen tissues fixed with acetone or formalin. Fresh frozen sections were fixed with ice-cold acetone for 30 min (A,C) or 10% formalin in phosphate buffer (pH 7.4) for 30 min (B,D). Then p300 was localized in the uterus (A,B) and claudin-1 was detected in the liver (C,D). Bar = 50 µm.

View larger version (119K): [in this window] [in a new window]   Figure 4 Effect of fixatives and heating on immunostaining of androgen receptor (AnR) and -amylase. AnR was localized in the head of epididymis (A–D) and -amylase was detected in the pancreas (E–H). Fresh frozen sections were fixed with 10% formalin in 0.1 M phosphate buffer (pH 7.4) for 30 min (A,B,E,F), or with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl2 for 30 min (C,D,G,H). The sections were then immunostained without heat treatment (A,C,E,G) or after autoclaving at 120C for 10 min in 20 mM Tris HCl buffer, pH 9.0 (B,D,F,H). Bar = 50 µm.

View larger version (105K): [in this window] [in a new window]   Figure 5 Localization of hormone-occupied and -unoccupied estrogen receptor (ER) and glucocorticoid receptor (GR) in frozen sections. To detect liganded and unliganded ER, immature female mice (2 weeks old) were intraperitoneally injected with 17ß-estradiol (20 µg/kg BW) (B,D) or vehicle solution (A,C) and sacrificed 1 hr after the injection. Fresh frozen sections were fixed with 10% formalin in 0.1 M PB (pH 7.4) for 30 min at room temperature (A,B), or with 0.5% glutaraldehyde in 0.1 M PB (pH 7.4) for 1 min at room temperature followed by autoclaving for 10 min in 20 mM TB, pH 9.0 (C,D). The sections were then immunostained with polyclonal ER antibody. To localize hormone-bound and hormone-free GR, adrenalectomized mice were intraperitoneally given 25 µg of dexamethasone (F,H) or vehicle solution (E,G), and killed after 1 hr. Frozen sections were fixed with 10% formalin in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (E,F), or fixed with 20% formalin containing 75 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.4) for 30 min at 40C and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) (G,H). Inserts in D and H show control immunostaining of ER and GR, respectively; antibodies preabsorbed with antigenic peptides were used as the primary antibodies. Bar = 25 µm.

View larger version (161K): [in this window] [in a new window]   Figure 3 Effect of fixatives and heating on proliferating cell nuclear antigen (PCNA) immunostaining. PCNA was localized in the mouse duodenum. Fresh frozen sections were fixed with 10% formalin in 0.1 M phosphate buffer (PB) (pH 7.4) for 30 min (A,D), with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl2 for 30 min (B,E), or with 0.5% glutaraldehyde in 0.1 M PB (pH 7.4) for 1 min (C,F) at room temperature. The sections were then immunostained without heat treatment (A–C) or after autoclaving at 120C for 10 min in 20 mM TB, pH 9.0 (D–F). Arrows indicate plasma cells (A). Bar = 50 µm.

When the aldehyde-fixed frozen sections from mice were immunostained with monoclonal antibodies (mouse IgG), heating completely diminished the immunostaining in the plasma cells, connective tissues, and blood vessels, which corresponds to the endogenous immunoglobulin (Figure 3). All controls for immunohistochemistry exhibited negative staining (Figure 1A; Figures 5D and 5H, insets).

Detection of Unliganded ER and GR in Aldehyde-fixed Fresh Frozen Sections
When frozen sections were fixed with 10% formalin for 10–30 min at room temperature or 40C, the epithelial cells of the uterus and oviduct of mice injected E2 showed stronger ER immunostaining than those of vehicle-injected controls (Figures 5A and 5B). When the frozen sections were fixed with 0.5% glutaraldehyde in 0.1 M PB (pH 7.4) for 1 min followed by autoclaving, these cells exhibited almost the same ER immunostaining intensity regardless of the hormonal status (Figures 5C and 5D), although the reaction was faint or negative without autoclaving. Two antibodies, a polyclonal and a monoclonal antibody, yielded almost the same patterns of HIAR in the glutaraldehyde-fixed fresh frozen sections (data not shown).

When tissues were fixed with 10% formalin for 30 min at room temperature or 40C, strong GR immunostaining was present in the nucleus of various cell-types in the liver, pancreas, and small intestine of adrenalectomized mice treated with dexamethasone (Figure 5F), whereas faint GR immunostaining was observed in the nucleus of vehicle-treated control animals (Figure 5E). In the sections fixed with 20% formalin containing 50 mM or 75 mM CaCl₂ at 40C for 30 min and then autoclaved, unliganded GR also showed strong nuclear staining and weak cytoplasmic staining, although the staining intensity in the nucleus was still weaker than that of liganded GR (Figures 5G and 5H).

Effect of EDTA on Immunostaining
To study the effect of calcium ions, fresh frozen sections fixed with formalin containing 25 mM CaCl₂ for 30 min were incubated with 20 mM TB (pH 9.0) with or without 1 mM EDTA at room temperature for 2 hr, or autoclaved in the same solutions for 10 min. Treatment with EDTA at room temperature or at 120C had no significant effect on the immunostaining patterns of all antigens (Figure 6), although the immunostaining of some antigens such as ER, p300, AnR, and integrin 3, decreased slightly when the sections were autoclaved in TB containing 1 mM EDTA.

View larger version (118K): [in this window] [in a new window]   Figure 6 Effects of EDTA on the immunostaining of the tissues fixed with formalin containing CaCl2 Fresh frozen sections were fixed with 10% formaldehyde containing 25 mM CaCl2 dissolved in 0.1 M cacodylate buffer (pH 7.4) for 30 min at room temperature. The sections were then immersed in 20 mM Tris-HCl buffer (TB) (pH 9.0) with (B,F,J) or without 1 mM EDTA (A,E,I) at room temperature for 2 hr, or autoclaved for 10 min in 20 mM TB (pH 9.0) (C,G,K) or in the buffer containing 1 mM EDTA (D,H,L). Then, steroid receptor coactivator-1 was immunolocalized in the oviduct (A–D) and claudin-5 was detected in the duodenum (E–H). Laminin-immunostaining was shown in the pancreas (I–L). In G and H, tight junctions immunostained with claudin-5 are seen in the endothelial cells of blood vessels (large arrows) and the glandular epithelium (small arrows). Bar = 25 µm.

	Discussion

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Formaldehyde solutions containing calcium ions, such as Baker's formol calcium, had been used for enzyme histochemistry; they are assumed to stabilize membrane phospholipids and to minimize diffusion of enzymes from cell organelles (Baker 1944; Bancroft 1996). In addition, we have demonstrated that calcium ions accelerate crosslinking of proteins in vitro (Yamashita and Okada 2005). However, they have not been used for immunohistochemical studies, probably because of the severe reduction or loss of immunoreaction. Morgan et al. (1994)(1997) assumed that coordinate bonds between calcium and the methyrol groups of proteins introduced by formaldehyde form a cagelike structure and prevent antigen–antibody interactions. The results of the present study showed that EDTA had no effect on the immunostaining pattern of any of the antigens, with or without heating, and suggested that the cagelike structure is not present in the tissues fixed with formaldehyde as reported by Shi et al. (1999). Our previous study in vitro also suggested that the proteins treated with formaldehyde do not form the tight cagelike structure with calcium ions (Yamashita and Okada 2005). Even if coordinate bonds are present between calcium ions and proteins, the bonds may be unstable than the bonds between calcium ions and EDTA, because polypeptides maintain their secondary and tertiary structure after formaldehyde fixation (Mason and O'Leary 1991) and may be not so flexible enough to form stable coordinate bonds with calcium ions.

It is well known that sex steroid receptors localize in the nucleus, regardless of hormonal status and that unliganded receptors are readily extracted from fresh frozen sections during fixation (Yamashita and Korach 1989; Slayden et al. 1995). Therefore, fixation procedure that yields similar staining intensity of liganded and unliganded ER is a good model system for evaluating the success of fixation. Liganded and unliganded ER exhibited almost the same staining intensity in the mouse uterine epithelial and stromal cells when fresh frozen sections were fixed with glutaraldehyde followed by autoclaving. By contrast, the subcellular localization of unliganded GR is still a matter of controversy in a variety of cell types in of tissues and cultured cells (Gasc et al. 1989; McGimsey et al. 1991; Pekki et al. 1992; Yamashita 2001). In the present study, ligand-free GR was barely detected in the nucleus in mouse tissues when fresh frozen sections were fixed with 10% or 20% formalin at room temperature. However, when the sections were fixed with 20% formalin containing 50 or 75 mM CaCl₂ at 40C, a strong immunoreaction for ligand-free GR was observed in the nucleus and weak reaction was present in the cytoplasm after HIAR, whereas the reaction of ligand-free GR in the nucleus was weaker than that of liganded GR. The present findings suggest that unliganded GR is present in both the nucleus and the cytoplasm of mouse tissues, and that one of the reasons of conflicting reports concerning localization of ligand-free GR is attributable to differences in fixation protocols.

The results of the present study demonstrate that HIAR is a powerful technique not only for formalin-fixed and paraffin-embedded specimens, but also for aldehyde-fixed fresh frozen tissues. The mechanisms of HIAR may be the same in paraffin sections and frozen sections. Heating cleaves intra- and intermolecular crosslinks and extend polypeptide chains and antibodies may be easily penetrate into the tissues. In addition, electrostatic repulsion by negatively charged polypeptides and hydrophobic attraction may balance to prevent intertwining of unfolded polypeptide chains in a retrieval solution at basic pH and antigenic determinants may be exposed to react with antibodies (Yamashita and Okada 2005; Emoto et al. 2005). Rapid and complete fixation procedure that minimizes diffusion artifacts, false localization, and extraction of antigens during fixation and heating is more important for fresh frozen sections than paraffin sections or frozen sections prepared from tissues prefixed with aldehyde. HIAR may also be a useful technique for immunoelectron microscopy of many antigens with the preembedding method, using prefixed frozen unfrozen or frozen sections (Yamashita et al. 1989).

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid (16590154) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

	Footnotes

 
Received for publication November 11, 2004; accepted June 23, 2005

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