Evaluation of procedures for the fixation and processing of human tissue for immunohistochemical analysis of human monoclonal antibodies

Abstract

Preliminary investigations suggested the importance of an evaluation of different tissue preparation methods frequently used for immunohistochemical analysis of human or murine monoclonal antibodies on human tissue. Colon adenocarcinomas and adjacent morphologically normal colon epithelia were analyzed with an indirect immunoperoxidase technique. Duplicate tissue specimens were (1) snap frozen and fixed in acetone, (2) formalin fixed and paraffin embedded, with or (3) without ensuing treatment with pronase, or (4) alcohol fixed and paraffin embedded. Three different human monoclonal anti-colon cancer IgM antibodies, COU-1, D4213, and F10279, were used in the present study. Endogenous immunoglobulin and the secretory-component-mediated IgG binding were blocked on frozen sections with Fab' anti-IgM and anti-SC antibody. Bound monoclonal antibody was detected with horseradish peroxidase-anti-IgM. COU-1 was found to stain frozen sections of all 25 cancer and adjacent normal colon epithelia. In contrast, on formalin-fixed, paraffin-embedded tissue, only 80% (20/25) of the colon cancer and 44% (11/25) of the adjacent normal colon epithelia were positive. After treatment of the formalin-fixed sections with pronase, all cancers and normal adjacent epithelia were stained, but the cancer cells were more intensely stained than the normal colon epithelial cells. On alcohol-fixed tissues, intense staining was found in all the colon carcinomas analyzed, whereas no staining was found of the adjacent normal colon epithelia, except for a few cells in some of the sections investigated.(ABSTRACT TRUNCATED AT 250 WORDS)