Nonspecific Nuclear Immunoreactivity After Antigen Retrieval Using Acidic and Basic Solutions

Abstract

Reports of false positive immunoreactivity after antigen retrieval (AR) have been rare. Since instituting AR in our laboratory in 1991, 10/371 (2.7%) of tissues exhibited nuclear positivity after AR while immunostaining for cytoplasmic antigens. Three of the 10 tissues (1 gastric carcinoma, 1 mycosis fungoides skin, and 1 chronic dermatitis) were studied in detail with and without AR with the V9 monoclonal antibody to vimentin, a ubiquitous antigen, and the polyclonal antibody to prostate specific antigen (PSA), not normally present in these tissues. AR was performed with 3 solutions: 1% zinc sulfate (pH 4.9), 0.01 M citrate (pH 6.0), and 0.01 M Tris (pH 9.0). A 3-step streptavidin-biotin peroxidase technique with multiple blockers, varied primary antibody concentration, and overnight primary incubation was performed.Results showed that nuclear immunoreactivity was augmented after AR at high concentrations of primary antibody, especially after AR with 1% zinc sulfate (pH 4.9). The nuclear staining was present in practically all nuclei and could not be blocked. A simple retitration to a more dilute primary antibody concentration (10- to 100-fold) abolished this nonspecific staining without affecting normal V9 or anti-PSA immunoreactivity (The J Histotechnol 20:139, 1997)