IHC Staining of Paraffin Sections

All paraffin-embedded tissues were sectioned at 4 μm in thickness for IHC staining, and the procedure was carried out as the manual of EnVision^™ Detection Systems Peroxidase/DAB (Dako, Denmark, #K5007). The epitope retrieval was performed by heat-induced technique. Briefly, tissue slides were deparaffinized in gradient xylene and ethanol, and then boiled in a pressure cooker with 0.01 M citrate buffer (pH 6.0) for 10 minutes. After cooling down to the room temperature, sections were treated by serum blocking reagent for 30 min. Primary antibody was then used for incubation at 4°C in a humidity chamber overnight, followed by the secondary antibody, DAB developer and hematoxylin staining application in turn. Commercially purchased IHC primary antibodies directed against the following antigens were: RPS6KB1 (Anti-RPS6KB1 E343 antibody, #ab32529), p-RPS6KB1 (Anti-RPS6KB1 phospho T389 antibody, #ab126818). The confirmed colon carcinoma specimens were used for positive controls, while PBS instead of the primary antibodies were negative controls [17 (link)].