OPTIMAL CONDITIONS OF FIXATION FOR IMMUNOHISTOCHEMICAL STAINING OF PROLIFERATING CELL NUCLEAR ANTIGEN IN TUMOR-CELLS AND ITS CELL-CYCLE RELATED IMMUNOHISTOCHEMICAL EXPRESSION

Comparison of the results of immunohistochemical expression, such as p roliferating cell nuclear antigen (PCNA) in archival material of tumou rs, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain st able and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fixation, proce ssing and staining of samples using animal-implanted MBT-2 cells deriv ed from chemical-induced mouse bladder carcinoma and PC10, a monoclona l antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temp erature before fixation for less than 12 h, fixation in formaldehyde w as less than 48 h, and paraffin-embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PC NA positive rates obtained under these conditions was compared with th e bromodeoxyuridine (BrdUrd) labelling indices. Although the average P CNA positive rate was significantly higher than the BrdUrd labelling i ndex (P less than or equal to 0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In ord er to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Oc currence of PCNA positive G, cells was expected at a half-life of the PCNA-protein of 20 h and a tumour cell doubling time of about 24 h. Th us, the percentage of PCNA positive nuclei in a conventionally paraffi n-embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of th e biological malignant potential of tumours.