title: An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections.
abstract: 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% OsO(4), 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.
authors.affiliation: Department of Pathology, Oita Medical University, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Yano
authors.forename: Kenji
authors.initials: K
authors.lastname: Kashima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Daa
authors.forename: Shogo
authors.initials: S
authors.lastname: Urabe
authors.forename: Koichi
authors.initials: K
authors.lastname: Tsuji
authors.forename: Iwao
authors.initials: I
authors.lastname: Nakayama
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000180:Acrylic Resins; D000310:Adrenal Gland Neoplasms / Q000378:metabolism / Q000648:ultrastructure; D002021:Buffers; D053379:Chromogranin A; D002864:Chromogranins / Q000378:metabolism; D004853:Epoxy Resins; D006361:Heating; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007340:Insulinoma / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron / Q000379:methods; D008867:Microtomy; D008872:Microwaves; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000648:ultrastructure; D010673:Pheochromocytoma / Q000378:metabolism / Q000648:ultrastructure; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D012996:Solutions; D016610:Tissue Embedding
chemical_list: D000180:Acrylic Resins; D002021:Buffers; D053379:Chromogranin A; D002864:Chromogranins; D004853:Epoxy Resins; D012996:Solutions; C048707:LR white