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Journal of Histochemistry and Cytochemistry, Vol. 51, 199-204, February 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

An Antigen Retrieval Method Using an Alkaline Solution Allows Immunoelectron Microscopic Identification of Secretory Granules in Conventional Epoxy-embedded Tissue Sections

Shinji Yanoa, Kenji Kashimaa, Tsutomu Daaa, Shogo Urabeb, Koichi Tsujib, Iwao Nakayamaa, and Shigeo Yokoyamaa
a Department of Pathology, Oita Medical University, Oita, Japan
b Central Laboratory, Oita Prefectural Hospital, Oita, Japan

Correspondence to: Kenji Kashima, Dept. of Pathology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan. E-mail: kkashima@oita-med.ac.jp

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO4, and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 ± 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 ± 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.

(J Histochem Cytochem 51:199–204, 2003)

Key Words: immunoelectron microscopy, chromogranin A, secretory granule, antigen retrieval, epoxy-embedded tissue


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