Iscan coreo slide scanner

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.

Synovial macrophages were identified with immunostaining techniques as previously described [25 (link)]. Briefly, 4 μm tissue sections were deparaffinized, hydrated in graded ethanol and incubated in 4% bovine serum albumin (BSA) and 3% sheep serum to block unspecific immunobinding. A monoclonal mouse anti-rabbit macrophage antibody (RAM11, 36.2 mg L^− 1, Dako, Glostrup, Denmark) was added overnight, 4 °C. The binding was detected using biotinylated goat anti-mouse immunoglobulin G (IgG) (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and peroxidase ABC with 3,3 diaminobenzidine tetra-hydrochloride as chromogen (Dako, Golstrup, Denmark). Sections were counterstained with hematoxylin, mounted in DPX medium (VWR International, Leuven, Belgium) and photographed using an automated iScan Coreo slide scanner (Ventana Medical Systems, USA). Five random areas per slide were selected for a blinded evaluation and quantified with Image J software as previously described [25 (link)]. Area selection relies on the following criteria: 1) both lining and sublining are well represented, 2) areas with blank spaces are excluded, 3) areas with artifacts are excluded. Results were expressed as the percentage of positive stained area.