YAP protein expression in human cervical tissues was detected by using peroxidase-based immunohistochemistry as described previously (Fu et al, 2014 (link)). Briefly, human tissues were deparaffinized with xylene, rehydrated with graded ethanol series, and then autoclaved in an unmasking solution (Vector Laboratories, Burlingame, CA) for antigen retrieval before blocking endogenous peroxidase activity with 3% hydrogen peroxide. Tissues were next blocked with 10% normal donkey serum (NDS) at room temperature for 1 h followed by incubation with primary antibodies at 4°C for 16 h. Biotinylated secondary antibody and streptavidin peroxidase complex (DAKO LSAB Kit, Carpinteria, USA) were added consecutively for ten minutes at room temperature. The immunosignal was visualized with DAB kit (Invitrogen, Carlsbad, CA). The sections were counterstained with Mayer’s hematoxylin. For negative controls, the primary antibody was replaced with blocking buffer containing the same amount of IgG from the non-immune rabbit serum. Sections were scanned with an iSCAN Coreo Slide Scanner (Ventana Medical Systems, Inc. Oro Valley, AZ). The intensity of the positive immunosignal was quantified using Aperio ImageScope software (Vista, CA). The intensity of positive signal and the positivity (i.e., the ratio of positive cell number relative to the total cell) of each section were recorded.
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