Copyright ©The Histochemical Society, Inc.


RAPID COMMUNICATION

Mechanisms of Heat-induced Antigen Retrieval : Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry

Shuji Yamashita and Yasunori Okada

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

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


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
In this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl2, then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter- and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of ß-actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes. (J Histochem Cytochem 53:13–21, 2005)

Key Words: antigen retrieval • SDS-PAGE • Western blot • immunohistochemistry • epitope conformations


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SEVERAL ANTIGEN RETRIEVAL/EXPOSURE PROCEDURES have been applied to immunohistochemistry in routinely processed pathological materials, such as formalin-fixed and paraffin-embedded tissues. Since its introduction by Shi et al. (1991)Go, heat treatment has become the most popular method (Werner et al. 1996Go; Pileri et al. 1997Go; Shi et al. 2001Go). The disruption of formaldehyde-induced cross-links is believed to play a major role, although the mechanisms of antigen retrieval are not fully understood (Shi et al. 1991Go; Cattoretti et al. 1993Go).

We previously demonstrated that some monoclonal antibodies are unable to react with native antigens but bind to antigens treated with high temperatures or denaturing reagents, such as urea or sodium dodecyl sulfate (SDS), not only in tissues but also in immunoblots (Yasuda et al. 1986Go; Yamashita et al. 1997Go). Pfund et al. (1996)Go also reported that a monoclonal antibody prepared against bovine somatotropin reacted only with the denatured antigen. These results suggest that epitopes located on the inner portions of antigen molecules or those hidden by other oligomeric components must be exposed through denaturation before they can react with antibodies, regardless of treatment of fixatives (Yasuda et al. 1986Go; Yamashita et al. 1989Go,1997Go; Robinson and Vandre 2001Go).

Methylene bridges are assumed to be the major structural element of cross-links, which may form in proteins and nucleic acids (Auerbach et al. 1977Go; Shi et al. 2001Go). Masuda et al. (1999)Go demonstrated that formaldehyde treatment added mono-methylol (-CH2OH) groups to oligo RNAs at various rates and formed intramolecular cross-links via methylene bridges. These investigators also reported that the majority of the methylol groups could be removed from the bases by heating in a formalin-free buffer at 70C for 1 hr. However, no direct evidence of the cleavage of the methylene bridges formed in proteins has been obtained except for the report of Rait et al. (2004)Go.

In this study we analyzed, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the polymerization, modification, and breakdown of several proteins that had been treated with formaldehyde and then heated under various conditions to elucidate the mechanisms of heat-induced antigen retrieval procedures. Three acidic proteins, i.e., bovine serum albumin (BSA), ovalbumin (OA), and soybean trypsin inhibitor (TI), and two basic proteins, i.e., egg white lysozyme (LY) and bovine pancreatic trypsin (TR), were examined. By using a Western blot technique, we examined the presence of immunoreactive polypeptides in extracts from paraffin sections after heating. In addition, immunohistochemistry of 10 antigens with various isoelectric points (pIs) was carried out to clarify the following problems: (a) the relationship between the pI of antigen proteins and the optimal pH of antigen retrieval solutions and (b) effects of pH on the conformation of epitopes.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Reagents
BSA (fraction V) and LY were purchased from Seikagaku Kogyo (Tokyo, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. OA (grade VI), TI (type 1-S), and TR (type X III) were from Sigma Chemical (St Louis, MO). Anti-estrogen receptor {alpha} (ER{alpha}) rabbit antibody (sc-542), anti-androgen receptor (AR) rabbit antibody (sc-815), anti-p300 rabbit antibody (sc-585), and anti-steroid receptor co-activator-1 (SRC-1) rabbit antibody (sc 6098) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Immunizing peptides for ER{alpha} and AR, sc-542P, and sc-815P were also from Santa Cruz Biotechnology. Anti-ERß rabbit antibody (PA1-310B) and the neutralizing peptide (PEP-007) and anti-glucocorticoid receptor (GR) rabbit antibody (PA1-511) were purchased from Affinity Bioreagents (Golden, CO). Rabbit antibodies to laminin (L 9393) and fibronectin (F 3648) were from Sigma. Anti-{alpha}-amylase rabbit antibody was prepared in our laboratory (Yamashita 1981Go). Antibody to ß-actin was kindly supplied by Dr. Yasuhiro Sakai (Department of Anatomy, Kitasato University). Labeled polymer, horseradish peroxidase (HRP) (Envision+ system) was purchased from DAKO (Carpinteria, CA). A blocking reagent and block ace were from Boehringer Mannheim (Mannheim, Germany) and Dainippon Pharmaceutical (Osaka, Japan), respectively. Enhanced chemiluminescence (ECL) Western blotting detection kit was purchased from Amersham Biosciences (Piscataway, NJ). Paraformaldehyde and precasted acrylamide gels (e-PAGEL) were from Taab Laboratory Equipment (Aldermaston, UK) and Atto Corporation (Tokyo, Japan), respectively. Coomassie Brilliant Blue R-250 was from Nakarai Tesque (Kyoto, Japan).

Analyses In Vitro Employing SDS-PAGE
Treatment of Proteins with Formaldehyde
Treatment of proteins mimicked the procedure of paraffin embedding: fixation, washing with buffers, and treatment with organic solvents. Proteins (10 mg/ml) dissolved in saline (0.85% NaCl) were mixed with an equal volume of 8% formaldehyde dissolved in 0.2 M phosphate buffer (pH 7.2) and incubated for 25 hr at room temperature. Formaldehyde solution was freshly prepared from paraformaldehyde. LY was also treated with the formaldehyde solution for 30 min. To examine the effects of calcium ion, the proteins in saline were incubated with an equal volume of 8% formaldehyde in 0.2 M cacodylate buffer (pH 7.2) with or without 50 mM CaCl2 for 5 hr at room temperature. These sample preparations were dialyzed against 10 mM Tris-HCl buffer (pH 6.0) overnight at room temperature. A 4-fold volume of ice-cold acetone was then added to the protein solution to precipitate the proteins, and the solution was left at –20C for 1 hr.

Heating and SDS-PAGE
The samples were centrifuged at 14,000 x g for 10 min at 4C. The precipitated proteins were then washed three times with ice-cold 80% acetone and dissolved or suspended in distilled water. The aliquots were then mixed with an equal volume of various kinds of buffers: 20 mM Tris-HCl buffer (pH 3.0, 6.0, 7.5 or 9.0); 20 mM citrate buffer (pH 6.0 or 7.5); and 20 mM phosphate buffer (pH 6.0, 7.5, or 9.0). EDTA (20 mM) was added to some of the buffers. The specimens were boiled in a water bath or microwaved in a domestic microwave oven at 500 W for 5, 15, and 30 min, respectively, or autoclaved for 10 or 20 min at 120C and then analyzed on SDS-PAGE according to the method described by Laemmli (1970)Go. The heated proteins were incubated with running buffer containing 2-mercaptoethanol for 1 hr at 37C and then centrifuged for 3 min using a microcentrifugator (Capsulefuge PML-060; Tomy Seiko, Tokyo, Japan). BSA and OA were run on 7.5% and 10% gels, respectively. TI, TR, and LY were analyzed on 12.5% gels. Native proteins were used as molecular weight markers: BSA (66 kD), OA (42.5 kD), TR (23 kD), TI (20 kD), and LY (14 kD). After electrophoresis, the proteins were stained with Coomassie Brilliant Blue R-250.

Immunohistochemical Procedures
Ten antigens were examined: six nuclear proteins, i.e., ER{alpha} (pI 8.3), ERß (pI 8.8), AR (pI 6.3), GR (pI 6.0), p300 (pI 8.8), and SRC-1 (pI 5.7); two extracelluar matrix proteins, i.e., laminin (pI 5.9) and fibronectin (pI 5.4); {alpha}-amylase (pI 6.5) and ß-actin (pI 5.2). The pI of each protein was calculated based on the database of Swiss-Prot and TrEMBL (protein knowledge database). CD-1 mice (8-week-old) were obtained from Clea Japan (Tokyo, Japan). Tissues from the mice were fixed with 4% formaldehyde dissolved in 0.1 M phosphate buffer (pH 7.2) for 6 hr at 4C, washed with phosphate-buffered saline (PBS) overnight at 4C, and then embedded in paraffin. Deparaffinized sections (4 µm) were boiled in 20 mM Tris-HCl buffers, pH 6.0 or pH 9.0, for 10 min in a domestic microwave oven and cooled for 1 hr at room temperature. The sections were briefly washed with distilled water and heated in another buffer (e.g., pH 9.0 and then pH 6.0) for 5 min and cooled in a manner similar to that described above. Some specimens treated in the second buffer were further heated in the first buffer (e.g., pH 9.0, then pH 6.0, and finally pH 9.0) for 5 min and allowed to cool. After washing with PBS, the sections were treated with a blocking solution, 1% BSA and 1% blocking reagent dissolved in PBS, for 1 hr at room temperature and then incubated with the antibodies overnight at 4C: anti-ER{alpha} (1:2500 dilution), anti-ERß (1:1000), anti-AR (1:500), anti-GR (1:500), anti-SRC-1 (1:500), anti-p300 (1:500), anti-laminin (1:1000), anti-fibronectin (1:5000), anti-{alpha}-amylase (1:5000), and anti-ß-actin antibodies (1:1000), all of which were diluted with the blocking solution. The sections were successively treated with labeled polymer HRP for 1 hr. For the controls, antibodies preabsorbed with immunizing peptides or normal rabbit IgG were used in place of specific antibodies. The imidazol-3, 3' diaminobenzidine tetrahydrochloride solution was used as a chromogen. Some sections were counterstained with hematoxylin. Immunostaining of ER{alpha}, p300, and SCR-1 was evaluated in the epithelial cells of the uterus. ERß and AR immunoreactions were examined in the granulosa cells of the ovary and epithelial cells of the epididymis, respectively. Immunostaining of GR and fibronectin was studied in the liver. Laminin and ß-actin were localized in the small intestine and {alpha}-amylase was detected in the exocrine pancreas.

Western Blotting Analyses of Extracts from Deparaffinized Sections
Deparaffinized sections from mouse uteri (4 µm) were autoclaved for 15 min in 20 mM Tris-HCl buffer, pH 7.5 or pH 9.0, or were left unheated; the sections mounted on eight slide glasses were used for each treatment. After cooling, the sections were incubated with 2% SDS, scraped away from the slide glasses, and homogenized with a microtube homogenizer. A 4-fold volume of cold acetone was added to each homogenate and samples were stored at –70C. After centrifugation, the precipitate was dissolved with 100 µl of running buffer containing 2% SDS, 8 M urea, and 5% 2-mercaptoethanol and kept overnight at 37C. After centrifugation, each supernatant was subjected to SDS-PAGE with a 7.5% gel, and separated polypeptides were then transblotted onto a polyvinylidene difluoride membrane. The membrane was treated with the block ace for 1 hr at room temperature and subsequently with the anti-ß-actin antibody (1:5000) or anti-fibronectin antibody (1:5000) overnight at 4C with gentle shaking. The membrane was washed with PBS containing 0.05% Tween-20 and incubated with the labeled polymer HRP (1:100) for 1 hr. The enzyme activity of HRP was detected using an ECL method.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Effects of Formaldehyde on Proteins
The BSA, OA, and TI solutions were clear even after 25 hr incubation with formaldehyde, although LY and TR solutions became turbid within 5 min after incubation with formaldehyde and formed precipitates after 30 min (Figure 1).



View larger version (81K):
[in this window]
[in a new window]
 
Figure 1

Proteins treated with formaldehyde for 25 hr. Proteins in saline (10 mg/ml) were mixed with an equal volume of 8% formaldehyde dissolved in 0.2 M phosphate buffer, pH 7.2, and incubated for 25 hr at room temperature. BSA, bovine serum albumin; OA, ovalbumin; TI, soybean trypsin inhibitor; TR, bovine pancreatic trypsin; LY, egg white lysozyme.

 
Monomers of BSA and OA treated with formaldehyde exhibited larger electrophoretic mobilities compared with untreated native proteins: apparent molecular weights of BSA and OA monomers were 50 kD and 35 kD, respectively (Figures 2A and 2B, Lanes 1 and 2). Dimers and trimers of these proteins treated with formaldehyde also migrated more rapidly than those of native proteins on SDS-PAGE. Changes in apparent molecular mass were not evident in TI monomer using the present electrophoresis system, although TI dimers and trimers treated with formaldehyde were ~5 kD smaller than those of TI treated with formaldehyde followed by heating (Figure 2C, Lanes 1 and 2).



View larger version (85K):
[in this window]
[in a new window]
 
Figure 2

Effects of heating procedures and pH on formaldehyde-induced cross-links. BSA (A), OA (B), and TI (C) solutions were mixed with 8% formaldehyde in 0.2 M phosphate buffer (pH 7.2) for 25 hr at room temperature. LY (D) was incubated with the formaldehyde solution for 30 min. After dialysis, the proteins were boiled in a water bath for 5 min (Lanes 3–5) or 30 min (Lanes 6–8), or autoclaved at 120C for 10 min (Lanes 9–11) in 10 mM HCl buffer, pH 3.0 (Lanes 3, 6, and 9), pH 6.0 (Lanes 4, 7, and 10), and pH 9.0 (Lanes 5, 8, and 11). They were then analyzed on SDS-PAGE. BSA and OA were run on 7.5% and 10% gels, respectively. TI and LY were analyzed on 12.5% gels. Lane 1 of each gel shows formaldehyde-untreated native proteins, and Lane 2 demonstrates the formaldehyde-treated proteins before heating. (A) m, native monomer (66 kD); d, dimer (132 kD); t, trimer (198 kD).(B) m, native monomer (42.5 kD); d, dimer (85 kD); t, trimer (127.5 kD). (C) m, native monomer (20 kD); d, dimer (40 kD); t, trimer (60 kD). (D) m, native monomer (14 kD); d, dimer (28 kD); t, trimer (42 kD).

 
Formaldehyde treatment generated polymers in all proteins examined. However, the ratio of polymers to monomers differed greatly among the proteins. BSA and OA yielded small amounts of dimers, trimers, and tetramers (Figures 2A and 2B, Lanes 1 and 2), whereas both TI and LY underwent polymerization. Precipitates of LY after formaldehyde treatment for 30 min or 25 hr were not dissolved in the running buffer and showed only faint bands (Figure 2D, Lane 2). In contrast, they were solubilized after heating and LY polymers (dimers–decamers) were recognizable on SDS-PAGE (Figure 2D, Lanes 3 and 4). TR treated with formaldehyde for 25 hr was also not dissolved in the running buffer (data not shown).

Effects of Heating on Formaldehyde-treated Proteins
When autoclaved for 10 min or boiled in a water bath for 30 min at the proper pH (described below), BSA, OA, TI, and LY exhibited almost the same electrophoregrams as the native proteins (Figure 2). Boiling in a water bath or in a microwave oven produced similar results (data not shown). The amount of TR decreased considerably, however, because of autolysis. Heating at all pH ranges exhibited similar effects on the migration of proteins. However, proteins heated drastically at pH close to their respective pIs tended to precipitate. BSA (pI 5.6) formed insoluble precipitates at pH 6.0 and showed almost no bands on SDS-PAGE (Figure 2A, Lane 10), whereas heating at pH 9.0 yielded a large amount of insoluble precipitates of LY (pI 9.3) (Figure 2D, Lanes 8 and 11) and TR (pI 8.7). OA (pI 5.2) and TI (pI 4.6) were soluble at pH 3.0 and 6.0, but a large portion of proteins were unable to migrate through the stacking gels (Figures 2B and 2C, Lanes 9 and 10). BSA was partially decomposed when heated at pH 3.0 (Figure 2A, Lanes 6 and 9). LY also showed a small amount of heat-induced degradation products at pH 3.0 and 6.0 (Figure 2D, Lanes 6, 9, and 10).

Effects of Calcium Ion on Cross-links
Formaldehyde solution containing CaCl2 promoted the polymerization of OA and TI (Figures 3A and 3B, Lanes 1 and 2) but not that of BSA (data not shown). A large portion of TR underwent autolysis, resulting a long smear from trimer (69 kD) to monomer (23 kD) (Figure 3D, Lanes 1 and 2). The effects of calcium ion on LY could not be analyzed because LY precipitates were insoluble in the running buffer. No apparent changes in the electrophoretic patterns of the proteins were observed when the proteins treated with CaCl2-containing formaldehyde solution were analyzed before (Figure 3, Lanes 2 and 3) or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA (Figure 3, Lanes 4–7).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 3

Effects of calcium ion on formaldehyde-induced cross-links. OA (A), TI (B), LY (C), and TR (D) solutions were mixed with 8% formaldehyde in 0.2 M cacodylate buffer, pH 7.2 (Lane 1) or with the fixative containing 50 mM CaCl2 (Lanes 2–7) for 5 hr. After removal of formaldehyde, the proteins were analyzed on SDS-PAGE without heating in the absence (Lanes 1 and 2) or the presence (Lane 3) of 10 mM EDTA. The samples were boiled for 15 min in 10 mM Tris-HCl buffer, pH 6.0, in the absence (Lane 4) or the presence (Lane 5) of 10 mM EDTA, or in 10 mM Tris-HCl buffer, pH 9.0 in the absence (Lane 6) or the presence (Lane 7) of 10 mM EDTA. In D, Lane 8 reveals native TR untreated with formaldehyde: m, native monomer (23 kD); d, dimer (46 kD); t, trimer (69 kD). In A–C: m, monomer of each native protein; d: dimer; t: trimer.

 
Western Blot Analyses of the Extracts from Sections
Anti-ß-actin antibody reacted with 42-, 64-, 84-, 120-, and 170-kD polypeptides and higher-molecular-weight polypeptides showing a smear in the extracts from sections autoclaved for 15 min in 20 mM Tris-HCl buffer, pH 7.5 (Figure 4A, Lane 2) or pH 9.0 (Figure 4A, Lane 3). However, no bands were detected with the antibody in the extracts from unheated sections (Figure 4A, Lane 1). Polypeptides located at separating gel boundaries reacted with antifibronectin antibody in the extracts from sections autoclaved in Tris-HCl buffer, pH 7.5 or 9.0 (Figure 4B, Lanes 2 and 3), whereas the extracts from non-autoclaved sections showed no reaction (Figure 4B, Lane 1). A 250-kD polypeptide was also stained weakly with anti-fibronectin antibody. In the extracts from autoclaved sections, more than 10 bands were recognized with protein staining, but not in the extracts from unheated sections (data not shown).



View larger version (53K):
[in this window]
[in a new window]
 
Figure 4

Western blot analyses of ß-actin and fibronectin in extracts from paraffin sections. Deparaffinized mouse uterine sections were autoclaved for 15 min in 20 mM Tris-HCl buffer, pH 7.5 or pH 9.0, or were left unheated. Proteins were then extracted from the sections using 2% SDS solution containing 8 M urea and 5% 2-mercaptoethanol, and then analyzed with Western blotting. ß-Actin (A) and fibronectin (B) were detected on the blots: extracts from unheated sections (Lanes 1), extracts from sections autoclaved for 15 min in 20 mM Tris-HCl buffer, pH 7.5 (Lanes 2), and pH 9.0 (Lanes 3). Arrows show the positions of standard proteins for molecular weight. Arrowheads indicate major bands detected with the antibodies. Arrowheads (A) indicate ß-actin monomer (42 kD), dimer (84 kD) and trimer (120 kD); arrowhead (B) shows fibronectin monomer (250 kD).

 
Immunohistochemical Study
Effects of heating in Tris-HCl buffer, pH 6.0 or pH 9.0, on immunoreaction of 10 antigens are summarized in Table 1. All antibodies except for anti-{alpha}-amylase antibody showed much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. All nuclear proteins examined in this study were strongly immunostained in the nuclei, independent of their pIs, when heated at pH 9.0 (Figures 5E and 5L), although most of them were negative or faintly immunostained without heating or after heating at pH 6.0 (Figures 5B and 5I). Immunoreaction of fibronectin along the basal lamina in the hepatic sinusoid was enhanced after heating at pH 9.0 but was decreased by heating at pH 6.0 (Figures 5S and 5P). Heat treatment, particularly at pH 6.0, weakened the laminin immunostaining along the basal lamina of intestinal epithelium (data not shown). Heating at pH 9.0 enhanced the immunostaining of ß-actin in the intestinal epithelium, while heat treatment at pH 6.0 and pH 9.0 yielded a similar intensity of {alpha}-amylase immunostaining (data not shown). All controls with peptide-absorbed antibodies or normal rabbit IgG exhibited negative immunostaining (Figure 5G, inset). Tris-HCl buffer and citrate buffer revealed almost the same immunostaining pattern at pH 6.0, although five antigens heated in citrate buffer showed a slightly stronger reaction than those heated in Tris-HCl buffer (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 1

pH-dependent antigen retrieval in mouse tissuesa

 


View larger version (132K):
[in this window]
[in a new window]
 
Figure 5

Effects of pH on the immunostaining. Mouse tissues fixed with 4% formaldehyde for 6 hr at 4C were embedded in paraffin. ERß was localized in the ovary (A–G): inset (G) shows the control immunostaining in which preabsorbed antibody with immunizing peptide was used as the primary antibody. AR was immunostained in the caput epididymis (H–N), and fibronectin was detected in the liver (O–U). Deparaffinized sections were immunostained without heat treatment (A,H,Q). Sections were boiled in 20 mM Tris-HCl buffers (pH 6.0) for 10 min (B,I,P) and successively heated in 20 mM Tris-HCl buffer (pH 9.0) for 5 min after cooling (C,J,Q). The sections were further heated in Tris-HCl buffer (pH 6.0) for 5 min (D,K,R). Conversely, sections were boiled in 20 mM Tris-HCl buffers (pH 9.0) for 10 min (E,L,S) and further heated at pH 6.0 for 5 min (F,M,T). The sections were boiled again in the Tris-HCl buffer (pH 9.0) for 5 min (G,N,U). Bar = 50 µm.

 
The second heating at pH 6.0 of the specimens first heated at pH 9.0 significantly decreased the immunostaining of all antigens except for {alpha}-amylase (Figures 5F, 5M, and 5T), and the third heating of the samples at pH 9.0 recovered strong immunoreactivity of the antigens (Figures 5G, 5N, and 5U). Conversely, when the tissue sections heated at pH 6.0 were boiled at pH 9.0, immunoreaction was intensified (Figures 5C, 5J, and 5Q), but a third heating at pH 6.0 weakened the immunostaining of most antigens (Figures 5D, 5K, and 5R). Immunoreaction of {alpha}-amylase was uninfluenced when tissues were subjected to heating in the same way (data not shown).


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SDS-PAGE is a useful technique for analyzing protein modifications induced by fixatives (Hopwood et al. 1989Go; Rait et al. 2004Go). Formaldehyde forms intra- and intermolecular cross-links in proteins and is believed to add methylol groups to the side chains of amino acids. Intermolecular cross-links resulted in protein oligomers/polymers. Intramolecular cross-links yielded more rapid migration of the proteins (smaller apparent molecular mass) on SDS-PAGE compared with unmodified native proteins, because they may prevent the protein molecule from extending in the presence of SDS (Hopwood et al. 1988Go; Rait et al. 2004Go). LY (pI 9.3) and TR (pI 8.3) were strongly precipitated within 30 min, possibly because of rapid polymerization and a shift in pI from basic pH to neutral pH resulting in the modification of primary amines, particularly lysine. Rait et al. (2004)Go also reported such a pI shift of formaldehyde-treated RNase A.

The present study showed that the cross-links are cleavable by heating at all pH ranges in the model system in vitro. Anti-ß-actin antibody recognized with 42-, 84-, 120-, and 170-kD polypeptides in the extracts from autoclaved sections, which probably correspond to monomer/tetramer of ß-actin. The seared band with higher molecular mass may be ß-actin polymer and/or conjugates of ß-actin and other proteins. Fibronectin was also solubilized from the tissues after autoclaving; a faint 250 kD-band may be fibronectin monomer. These results demonstrate that heating can also break the intermolecular cross-links in tissues that are composed of highly concentrated and heterogeneous macromolecules, and that the solubilized proteins maintain antigenic activities.

In formaldehyde-fixed and paraffin-embedded tissues, epitopes may be directly modified by the formation of intra- and intermolecular cross-links and the simple addition of mono-methylol groups to the side chains of amino acids. Sompuram et al. (2004)Go have recently reported that by employing a model system with peptide antigens covalently bound to slide glasses, peptide antigens can be classified into three groups on the basis of their sensitivity to formalin fixation: group 1 peptides, very sensitive to formalin; group 2 peptides, susceptible to formalin fixation only when neighboring proteins were co-immobilized adjacent to the peptides; group 3 peptides, insensitive regardless of the presence of other neighboring proteins. These investigators also showed that antigenicities of polypeptides belonging to groups 1 and 2 were retrieved by heating. Intermolecular cross-links between macromolecules, including proteins and nucleic acids, which form a gel-like structure, should severely limit the penetration of antibodies into tissue sections. Therefore, heat-induced antigen retrieval procedures are considered to facilitate the interaction between antigens and antibodies by cleaving the cross-links. In addition, heating should also expose epitopes that are covered with oligomeric subunits or are located on the inner portion of the antigens regardless of formaldehyde fixation (Yasuda et al. 1986Go; Pfund et al. 1996Go; Yamashita et al. 1997Go).

The secondary and tertiary structures of epitopes disrupted by heating are probably renatured in solutions with suitable pH, buffer, and salt concentrations. It is well known that the pH of an antigen retrieval solution markedly influences the immunostaining intensity. In the present study, heating at pH 9.0 provided much stronger immunostaining than that at pH 6.0 in all antigens except for {alpha}-amylase, in agreement with previous data (Shi et al. 1996Go; Pileri et al. 1997Go). There was no direct relationship between the suitable pH for antigen retrieval and the pI of antigen molecules. Intensity of immunoreaction obtained by heating in a buffer was reversibly changed by successive heating in another buffer with a different pH. These findings suggest that the pH of the buffers is an essential factor for proper refolding of epitopes to react with antibodies. Degradation or extraction of epitopes appeared to be a minor factor at neutral or slight basic pH.

Many reports have demonstrated that unfolded polypeptides readily self-associate or randomly associate with other proteins. Whether an unfolded polypeptide takes a native conformation or forms random aggregations should be delicately balanced between hydrophobic and ionic forces (Stempfer et al. 1996Go; Hanson and Gellman 1998Go). The presence of salts decreases ionic interactions and subsequently increases hydrophobic association of unfolded polypeptides. Heating in 20 mM Tris-HCl buffer, pH 9.0, was effective for antigen retrieval of most antigens. Around pH 9.0, the hydrophobic force and the ionic repulsion force of negatively charged proteins may balance to prevent intertwining of unfolded polypeptide chains and to maintain a suitable conformation of many proteins in tissues. Itoh et al. (1995)Go reported that immunostaining of some antigens in ethanol-fixed tissues is intensified by heating. Ethanol treatment without formaldehyde fixation may disturb protein conformation and cause coagulation, because it breaks hydrogen bonds and exposes hydrophobic portions of proteins. Heating may partially reset the conformation of ethanol-fixed proteins.

Morgan et al. (1994)Go(1997Go) hypothesized that tight cage-like complexes between calcium ions and methylol groups in proteins mask antigens and that high temperatures and calcium chelators are needed to break the complexes. The present study indicated that formaldehyde solution containing calcium ion accelerates intermolecular cross-linking in TI and OA but not in BSA; calcium ion may chelate acidic proteins to facilitate polymerization. The effects of the calcium ion were unclear in LY and TR because a large amount of insoluble precipitate was formed by the formaldehyde treatment. However, EDTA did not affect the electrophoregrams of proteins treated with formaldehyde containing calcium ions when the proteins were analyzed without heating or after heating in Tris-HCl buffer at pH 6.0 or pH 9.0. These results suggest that calcium ion does not form tight intermolecular cross-links with methylol groups of formaldehyde-treated proteins (Shi et al. 1999Go), as reported by Morgan et al., or does not do so at least in the presence of SDS.


    Acknowledgments
 
We thank Drs Takayuki Shiomi and Sanae Mochizuki (Department of Pathology, Keio University) for critically reading the manuscript.


    Footnotes
 
Received for publication March 22, 2004; accepted June 15, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Auerbach C, Moutschen-Dahmen M, Moutschen J (1977) Genetic and cytogenetical effects of formaldehyde and related compounds. Mutat Res 39:317–361[Medline]

Cattoretti G, Pileri S, Parravicini C, Becker MH, Poggi S, Bifulco C, Key G, et al. (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83–98[Medline]

Hanson PE, Gellman SH (1998) Mechanistic comparison of artificial-chaperone-assisted and unassociated refolding of urea-denatured carbonic anhydrase B. Fold Des 3:457–468[Medline]

Hopwood D, Slidders W, Yeaman GR (1989) Tissue fixation with phenol-formaldehyde for routine histopathology. Histochem J 21:228–234[Medline]

Hopwood D, Yeaman G, Milne G (1988) Differentiating the effects of microwave and heat on tissue proteins and their crosslinking by formaldehyde. Histochem J 20:341–346[Medline]

Itoh H, Miyajima Y, Osamura RY (1995) Immunohistochemistry of intranuclear antigens. Jpn J Breast Cancer 10:3–10

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[Medline]

Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K (1999) Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res 27:4436–4443[Abstract/Free Full Text]

Morgan JM, Navabi H, Jasani B (1997) Role of calcium chelation in high-temperature antigen retrieval at different pH values. J Pathol 182:233–237[CrossRef][Medline]

Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174:301–307[Medline]

Pfund WP, Bourdage JS, Farley KA (1996) Structural analysis of bovine somatotropin using monoclonal antibodies and the conformation-sensitive immunoassay. J Biol Chem 271:14055–14061[Abstract/Free Full Text]

Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, et al. (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123[CrossRef][Medline]

Rait VK, O'Leary TJ, Mason JT (2004) Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-Structural and functional alterations. Lab Invest 84:292–299[CrossRef][Medline]

Robinson JM, Vandre DD (2001) Antigen retrieval in cells and tissues: enhancement with sodium dodecyl sulfate. Histochem Cell Biol 116:119–130[Medline]

Shi SR, Cote RJ, Hawes D, Thu S, Shi Y, Young LL, Taylor CR (1999) Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal? J Histochem Cytochem 47:463–470[Abstract/Free Full Text]

Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49:931–937[Abstract/Free Full Text]

Shi SR, Cote RJ, Young L, Ashraf SA, Taylor CR (1996) Use of pH 9.5 Tris-HCl buffer containing 5% urea for antigen retrieval immunohistochemistry. Biotech Histochem 71:190–196[Medline]

Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748[Abstract]

Sompuram SR, Vani K, Messana E, Bogen SA (2004) A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol 121:190–199[CrossRef][Medline]

Stempfer G, Holl-Neugebauer B, Rudolph R (1996) Improved refolding of an immobilized fusion protein. Nat Biotechnol 14:329–334[Medline]

Werner M, Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253–260[CrossRef][Medline]

Yamashita S (1981) Immunohistochemical study of amylase and deoxyribonuclease in rat parotid gland. Acta Histochem Cytochem 14:236–260

Yamashita S, Aiso S, Shiozawa M, Yasuda K (1989) Immunohistochemical study of gamma-glutamyl transpeptidase with monoclonal antibodies. II. An immunoelectron microscopic study in rat kidney. Acta Histochem Cytochem 22:367–374

Yamashita S, Sogo T, Shiozawa M, Yasuda K (1997) Immunolocalization of aldolase A subunit using monoclonal antibody in rabbit tissues. Acta Histochem Cytochem 30:601–608

Yasuda K, Yamashita S, Aiso S, Shiozawa M, Komatsu T (1986) Immunohistochemical study of gamma-glutamyl transpeptidase with monoclonal antibodies. I. Preparation and characteristics of monoclonal antibodies to gamma-glutamyl transpeptidase. Acta Histochem Cytochem 19:589–600