Influence of formalin fixation time and tissue processing method on immunoreactivity of monoclonal antibody PC10 for...

Proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase-delta, has recently been proposed as a marker of proliferation that is detectable in formalin-fixed paraffin-embedded tissue. Fixation time has been known to influence protein immunoreactivity and therefore can significantly influence the results of a quantitative immunohistochemical assay. In this study, we investigate the relationship between formalin fixation time and immunoreactivity for PCNA in paraffin-embedded sections and examine the effect of postfixation tissue treatment with modified Bouin's solution. Samples of small and large intestine from two freshly sacrificed rats were fixed in 10% buffered formalin for 6, 30, 54, 174, 340, and 508 h. Standard histological processing was performed on paired specimens whose treatment differed only by predehydration immersion in a picric acid and mercuric chloride-containing solution. Paraffin sections were reacted with monoclonal antibody PC10 in a standard immunoperoxidase assay. Staining intensity for PCNA was scored on a scale of 0 to 10, and the mean number of PCNA-positive cells per crypt (10 crypts counted) was determined. No difference between animals was found. PCNA immunoreactivity was maximal in specimens fixed for 6 to 30 h, exponentially declining with longer fixation time. The rate of decline was mitigated in the treated sections. Fixation-time dependence of PCNA immunoreactivity has immediate implications for intra- and interlaboratory comparisons, especially in experimental studies in which specimens can be stored in formalin for variable times followed by batch processing. With regard to surgical pathology specimens, this study suggests that sample comparisons are valid, since routine fixation time is within the optimal rage for PCNA immunodetection.