title: Double autoimmunostaining with glycine treatment.
abstract: Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase-hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or NovaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.
authors.affiliation: The Second Department of Anatomy, Center of Chronic Viral Diseases, Kagoshima University Faculty of Medicine, Kagoshima, Japan. anahasui@m3.kufm.kagoshima-u.ac.jp
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Hasui
authors.forename: Tomio
authors.initials: T
authors.lastname: Takatsuka
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sakamoto
authors.forename: Sachie
authors.initials: S
authors.lastname: Matsushita
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Tsuyama
authors.forename: Shuji
authors.initials: S
authors.lastname: Izumo
authors.forename: Fusayoshi
authors.initials: F
authors.lastname: Murata
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology; D004396:Coloring Agents; D003713:Dendritic Cells / Q000166:cytology; D005998:Glycine; D006735:Horseradish Peroxidase; D006801:Humans; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D006965:Hyperplasia; D007150:Immunohistochemistry / Q000379:methods*; D007202:Indicators and Reagents; D008221:Lymphoid Tissue / Q000166:cytology; D012996:Solutions; D013601:T-Lymphocytes / Q000166:cytology; D014059:Tongue / Q000473:pathology
chemical_list: D004396:Coloring Agents; D007202:Indicators and Reagents; D012996:Solutions; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D005998:Glycine