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.
Pelosi
G
G
Servizio di Anatomia Patologica, Ospedale Civile Maggiore, Verona, Italy.
Bresaola
E
E
Menacherry
M J
MJ
Manfrin
E
E
Iannucci
A
A
D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology*; D002455:Cell Division; D002498:Centrifugation / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D009687:Nuclear Proteins / Q000032:analysis*; D018809:Proliferating Cell Nuclear Antigen; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen