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 degrees C followed by methanol at 20 degrees 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 -20 degrees C. In conclusion, our report indicates that PCNA is a labile antigen, which may critically be affected by temperature and air-drying procedures.