Scanning microfluorometric analysis of proliferating cell nuclear antigen in formalin-fixed sections of hyperplastic...

Scanning laser cytometric analysis of fluorochrome-labeled cells was used to survey and quantitate the distribution of proliferating cells in formalin-fixed, paraffin-embedded tissue sections from hyperplastic and neoplastic rat livers. The technique used fluorescent immunochemical staining of proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells and propidium iodide as a fluorescent nuclear counterstain. Use of an antigen retrieval treatment improved detection of PCNA and treatment of tissue sections with crystal violet improved the sensitivity of the method by quenching background autofluorescence. PCNA evaluation of cell proliferation in regenerating rat liver 0-48 h post-partial hepatectomy showed that 3-43% of cells stained positively for PCNA, a pattern closely correlating with previously reported rates of maximum DNA synthesis. The PCNA staining patterns observed among cells in neoplastic nodules were more focal in distribution and indicated that from 5 to 25% of the cell nuclei per nodular region stained positively for PCNA. This use of image analysis for the rapid identification of proliferating cell areas in fixed, paraffin-embedded tissue active in neoplastic growth will expedite in situ cytochemical and molecular studies attempting to identify key differences between hyperplastic and neoplastic growth.