Improved 1-h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital.
Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H and E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.
Kumada
Tokimasa
T
Department of Pathology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan.
Tsuneyama
Koichi
K
Hatta
Hideki
H
Ishizawa
Shin
S
Takano
Yasuo
Y
D000911:Antibodies, Monoclonal / Q000276:immunology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D046152:Gastrointestinal Stromal Tumors / Q000378:metabolism / Q000473:pathology; D006785:Hospitals, University; D006801:Humans; D007150:Immunohistochemistry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D007564:Japan; D053555:Keratin-5; D007633:Keratins / Q000032:analysis / Q000276:immunology; D008872:Microwaves; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000276:immunology; D015203:Reproducibility of Results; D012577:Schools, Medical; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods / Q000592:standards; D013997:Time Factors
D000911:Antibodies, Monoclonal; C503940:KRT5 protein, human; D053555:Keratin-5; D007633:Keratins; D019009:Proto-Oncogene Proteins c-kit