Methodological aspects of the immunostaining of proliferating cell nuclear antigen (PCNA) in cytospin preparations of MCF‐7 cell line

Cytospins of a human breast cancer cell line (MCF‐7) were studied for the expression of PCNA, a cell cycle‐related protein, using a variety of fixation and immunostaining procedures. The best fixative for PCNA was found to be buffered formaldehyde solution at 4°C followed by methanol at 20°C, whereas alcoholic fixatives decreased greatly the PCNA immunoreactivity. Air‐drying procedures of cytospins prior to and after fixation greatly undermined the PCNA immunostaining. A modified immunoperoxidase method provided a stronger staining of the PCNA‐reactive cells than the alkaline phosphatase anti‐alkaline phosphatase (APAAP) technique. PCNA immunoreactivity could be maintained up to 2 mo, putting slides in methanol at − 2°C. In conclusion, our report indicates that PCNA is a labile antigen, which may critically be affected by temperature and air‐drying procedures. © 1994 Wiley‐Liss, Inc.