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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

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

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