title: Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.
abstract: A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.
authors.affiliation: Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Nagahito
authors.initials: N
authors.lastname: Saito
authors.forename: Kohei
authors.initials: K
authors.lastname: Konishi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeda
authors.forename: Mototsugu
authors.initials: M
authors.lastname: Kato
authors.forename: Toshiro
authors.initials: T
authors.lastname: Sugiyama
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asaka
mesh_terms: D000180:Acrylic Resins; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology; D006361:Heating; D016480:Helicobacter pylori / Q000276:immunology; D006801:Humans; D016253:Microscopy, Immunoelectron / Q000379:methods*; D012996:Solutions; D013270:Stomach / Q000382:microbiology; D013696:Temperature; D013997:Time Factors; D016610:Tissue Embedding
chemical_list: D000180:Acrylic Resins; D000942:Antigens, Bacterial; D012996:Solutions; C037883:Lowicryl 4KM resin