DEVELOPMENT OF AN OPTIMAL PROTOCOL FOR ANTIGEN RETRIEVAL - A TEST BATTERY APPROACH EXEMPLIFIED WITH REFERENCE TO THE STAINING OF RETINOBLASTOMA PROTEIN (PRB) IN FORMALIN-FIXED PARAFFIN SECTIONS

The retinoblastoma (RE) gene, which encodes the nuclear RE protein (pR B), is believed to be involved in cell cycle control and cell differen tiation. Studies have demonstrated that loss of RE function may play a role in tumour formation and progression of a variety of human tumour s, such as bladder, lung, breast, and prostate cancers. The immunohist ochemical detection of pRB expression in formalin-paraffin sections of human cancer has potential advantages of convenience, economy, and co mpatibility with routine surgical pathology practice. In practice, how ever, results using PRE antibodies on routinely processed, paraffin-em bedded tissue have been inconsistent. In this study, the antigen retri eval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a 'test battery' approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solut ions at pH 1, 6, and 10 with three different heating conditions (tempe ratures 120 degrees C, 100 degrees C, and 90 degrees C). In the exampl e described here with antibody RB-WL-1, the low pH solution with the m icrowave heating at 100 degrees C proved most effective. Both fresh an d routinely processed formalin-paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining re sult on routinely processed formalin-paraffin sections with frozen sec tions of the same tumour. A consistent intensity of immunohistochemica l staining for pRB was achieved using the identified optimal AR protoc ol on formalin-paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pR B localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on p araffin sections was slightly to moderately stronger than that obtaine d in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bla dder carcinoma, with consistent pRB immunostaining results. The protoc ol described is simple to perform and gives reproducible results for e valuation of pRB expression by immunohistochemistry.