Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: heat-induced antigen retrieval fresh frozen section unoccupied steroid hormone receptors immunohistochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 1360 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 5100 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
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 1030 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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 antigenantibody 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 |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker JR (1944) Structure and chemical composition of the Golgi element. Q J Micr Sci 85:171
Bancroft JD (1996) Enzyme histochemistry. In Bancroft JD, Stevens A, Turner DR, eds. Theory and Practice of Histological Techniques. New York, Churchill Livingstone, 391410
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 171:8398[Medline]
Emoto K, Yamashita S, Okada Y (2005) Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens? J Histochem Cytochem (Epub ahead of print)
Evers P, Uylings HB (1997) An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods 72:197207[CrossRef][Medline]
Evers P, Uylings HB, Suurmeijer AJ (1998) Antigen retrieval in formaldehyde-fixed human brain tissue. Methods 15:133140[CrossRef][Medline]
Gasc JM, Delahaye F, Baulieu EE (1989) Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study. Exp Cell Res 181:492504[CrossRef][Medline]
Ino H (2003) Antigen retrieval by heating en bloc for pre-fixed frozen material. J Histochem Cytochem 51:9951003
Mason JT, O'Leary TJ (1991) Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 39:225229
McGimsey WC, Cidlowski JA, Stumpf WE, Sar M (1991) Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies. Endocrinology 129:30643072[Abstract]
Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, et al. (1995) ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Invest 73:149156[Medline]
Mighell AJ, Robinson PA, Hume WJ (1995) Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval. J Histochem Cytochem 43:11071114
Morgan JM, Navabi H, Jasani B (1997) Role of calcium chelation in high-temperature antigen retrieval at different pH values. J Pathol 182:233237[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 174:301307[CrossRef][Medline]
Pekki A, Koistinaho J, Ylikomi T, Vilja P, Westphal H, Touhimaa P (1992) Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique. J Steroid Biochem Mol Biol 41:753756[CrossRef][Medline]
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 183:116123[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 84:292299[CrossRef][Medline]
Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41:15991604
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 47:463470
Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49:931937
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 39:741748
Slayden OD, Koji T, Brenner RM (1995) Microwave stabilization enhances immunocytochemical detection of estrogen receptor in frozen sections of macaque oviduct. Endocrinology. 136:40124021[Abstract]
Werner M, Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253260[CrossRef][Medline]
Yamashita S (2001) Histochemistry and cytochemistry of nuclear receptors. Prog Histochem Cytochem 36:91176[Medline]
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 22:367374
Yamashita S, Korach KS (1989) Immunological analysis of the biochemical properties of the uterine estrogen receptor. Biol Reprod 40:12751285
Yamashita S, Okada Y (2005) Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry. J Histochem Cytochem 53:1321
Yamashita S, Sogo T, Shiozawa M, Yasuda K (1997) Immunolocalization of aldolase A subunit using monoclonal antibody in rabbit tissues. Acta Histochem Cytochem 30:601608
Yamashita S, Yasuda K (1992) Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion. J Histochem Cytochem 40:793806
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 19:589600
|