Ihc deparaffinization protocol

Immunohistochemistry was performed on resected tumor samples fixed in formalin by embedding with paraffin and sectioning the paraffin block (5 μm slices). Embedding, sectioning, and Ki67 proliferation staining were done by the MUSC Histology and Immunohistochemistry Shared Resource. Formalin-fixed paraffin-embedded (FFPE) tumor slices were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water (Abcam IHC deparaffinization protocol). According to the manufacturer’s instructions, antigens were demasked using a citric-acid-based unmasking solution (Vector Laboratories # H-3300-250). Sectioned slides were blocked using 0.1% saponin and 5% BSA prepared in 1X PBS for 1 h at room temperature. Slides were washed with 1X PBS before adding 1:100 primary antibody in BSA solution (0.1% saponin, 5% BSA, 1X PBS), shaking overnight at 4°C in a humidity chamber. Slides were again washed with 1X PBS before adding 1:500 secondary Alexa Fluor antibody (Jackson ImmunoResearch) for 1 h at room temperature in the dark (in a humidity chamber). Slides were mounted, and nuclei were stained (Vectashield Antifade Mounting Media with DAPI #H-1200-10) before imaging on the Olympus FV10i (Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313).