Retinoblastoma Susceptibility Gene Product Retrieval in Formalin-fixed, Paraffin-embedded Tissue: A Heating Method for Enhancing Immunohistochemical Staining

Abstract

We examined immunohistochemical methods to visualize the retinoblastoma susceptibility gene product (RB) in formalin-fixed, paraffin-embedded sections of the U2-OS and Saos-2 human osteosarcoma cell lines. Immunocytochemical studies showed clear staining with an anti-RB antibody in 88% of the smeared U2-OS cells and in none of the smeared Saos-2 cells. Antigen retrieval methods evaluated included microwaveoven and autoclave pretreatment of paraffin-embedded sections immersed in Dulbecco's phosphate buffered saline (pH 7.4) prior to incubation with the primary antibody. Strong and discrete staining of U2-OS cell nuclei for RB was seen after autoclave treatment, stronger than that seen following microwave-oven heating. Moreover, neither microwave-oven nor autoclave pretreatment produced positive staining in the nuclei of Saos-2 cells. Therefore, tissues previously considered unsuitable for immunohistochemical analysis of the RB can be studied after autoclave treatment.