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Journal of Histochemistry and Cytochemistry, Vol. 51, 1169-1176, September 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

Double Autoimmunostaining with Glycine Treatment

Kazuhisa Hasuia, Tomio Takatsukaa, Ryoichi Sakamotoa, Sachie Matsushitaa, Shin-ichiro Tsuyamaa, Shuji Izumob, and Fusayoshi Murataa
a The Second Department of Anatomy, Center of Chronic Viral Diseases, Kagoshima University Faculty of Medicine, Kagoshima, Japan
b Division of Molecular Pathology and Genetic Epidemiology, Center of Chronic Viral Diseases, Kagoshima University Faculty of Medicine, Kagoshima, Japan

Correspondence to: Kazuhisa Hasui, The Second Department of Anatomy, Kagoshima University Faculty of Medicine, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan. E-mail: anahasui@m3.kufm.kagoshima-u.ac.jp

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. (J Histochem Cytochem 51:1169–1176, 2003)

Key Words: autoimmunostaining, double immunostaining, glycine treatment, enzyme-labeled polymer, method (EnVision system), peroxidase–hydrogen peroxide, reaction dyes, mounting method, paraffin section, human tissue


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