Immunohistochemical visualization of histone H1 phosphorylation in squamous intraepithelial lesions of the gynecologic tract

Immunohistochemical staining was performed on gynecologic tract squamous intraepithelial lesions using a novel phosphorylation-specific monoclonal antibody (designated 12D11) that detects histone H1 when phosphorylated at a cyclin-dependent kinase (CDK)-responsive epitope. Findings were compared to immunostaining by MIB-1, an extensively studied antibody probe of proliferation. Routinely fixed and processed archival sections were subjected to distinct antigen retrieval and staining protocols for each antibody and were processed for immunodetection of either Ki-67 (with MIB-1) or phosphohistone H1, using a streptavidin-biotin kit and diaminobenzidine as chromagen. For 12D11 staining, antigen retrieval was performed at pH 4.0, and the antibody incubation buffer was supplemented with 1.0 M NaCl. Both 12D11 and MIB-1 stained parabasal cells in normal squamous epithelium. Staining by 12D11 and MIB-1 of cells in progressively higher strata was found to correlate with the severity of lesions. The mean proportion of positively stained cells was higher in MIB-1-stained sections than in 12D11-stained sections in normal squamous epithelium and in all grades of squamous intraepithelial lesions. We conclude that the changes in expression patterns of CDK-phosphorylated histone H1 in the spectrum of gynecologic squamous intraepithelial lesions are similar to staining patterns obtained with the proliferation probe MIB-1. The differing proportion of cells stained by MIB-1 and 12D11 suggests that phosphohistone H1 may be a useful alternative proliferation marker that detects a different subpopulation of cycling cells in premalignant squamous lesions.