AbstractThe effects of fixation procedures on the immunoreactivity of PCNA were investigated in smear preparations of HeLa S3 cells. The cells were fixed with acetone, methanol at 4°C, or both, and immunostaining was performed with a monoclonal antibody against PCNA (19A2). Although we failed to detect PCNA with either fixative alone or with a mixture of the 2 fixatives, 15 min fixation with acetone followed by 15 min fixation with methanol at 4°C yielded excellent nuclear staining. First, the PCNA labeling index (LI) was measured and compared with that of BrdU in smear preparations of HeLa cells fixed with acetone followed by methanol. The PCNA LI values (34.7±1.2% , n±10) we every similar to those of BrdU (36.8±0.8% , n = 10). Double immunostaining of PCNA/BrdU was then performed on HeLa cell smear preparations fixed by the same procedure. We observed cells positive for both antibodies with similar frequency of the labeling index values for 2 parameters in the smears. In contrast, there were no cells positive for either parameter alone. These results strongly suggest that, with 15 min fixation with acetone followed by 15 min fixation with methanol at 4°C, PCNA detection is specific for S phase cells. (The J Histotechnol17:325, 1994)