Improved 1-h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital

Abstract

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&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.

Expression profiling of endogenous secretory receptor for advanced glycation end products in human organs

“…The method employed was the tissue microarray technique. 4,5 This immunostaining method ensures a reproducible comparison among a variety of tissues from one individual or the same tissues from different individuals, which were aligned on the same slide. Tissue or cellular staining for esRAGE was classified into four different patterns, which might reflect distinct functional roles of this decoy receptor in different organs.…”

“…Immunostaining procedures were based on a new microwave technique as described previously. 5 In brief, the sections were deparaffinized, dehydrated, and treated for antigen retrieval with TRS-buffer solution (TRS; DAKO, CA, USA) in a wet chamber in a microwave oven (Type RE-11, Sharp, Tokyo, Japan; maximum 500 W) for 15 min. The sections were allowed to cool at room temperature for 30 min, rinsed under running water for 1 min, and then sequentially treated with 3% H 2 O 2 for 5 min to exhaust endogenous peroxidase.…”

Effects of Parafibromin Expression on the Phenotypes and Relevant Mechanisms in the DLD-1 Colon Carcinoma Cell Line

“…The immunohistochemistry was performed according to the procedures recommended as previously described (Kumada et al, 2004). The mouse anti-parafibromin or rabbit anti-ki-67 antibody were purchased from Santa cruz and DAKO respectively.…”

Liver‐targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis†

“…To investigate the distribution and frequency of cells expressing FoxP3 and the ratio of CD8 ϩ to CD3 ϩ cells, single immunoenzymatic staining was performed on sections of liver tissues from each patient using the FoxP3 (ab2481; Abcam, Ltd, Cambridge, UK), CD3 (monoclonal antibody; DAKO, Carpinteria, CA) and CD8 (monoclonal antibody; DAKO) antibodies and the microwave procedure (see supplemental material for FoxP3 specificity at the HEPA-TOLOGY website: www.interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html). 26 In brief, after deparaffinization and standard antigen retrieval via microwave irradiation, all sections were soaked in 3% H 2 O 2 for 5 minutes to inhibit endogenous peroxidase, rinsed, and incubated with 5% bovine serum albumin (bovine serum albumin, Sigma Chemical Co.) for 5 minutes to prevent nonspecific staining. Goat polyclonal anti-FoxP3 antibody (Abcam) diluted to 1/50, mouse monoclonal anti-CD3 (DAKO) or anti-CD8 (DAKO) antibodies diluted to 1/200 as mentioned above were respectively applied to the specimens in a plastic moist chamber for 15 minutes under intermittent microwave irradiation (MI-77, Azuyama, Tokyo, Japan; 250 W, 4 s on, 3 s off) followed by incubation at room temperature for an additional 45 minutes.…”