C kit antibody

Formalin-fixed paraffin embedded Sturge–Weber tissue was obtained from the Pediatric Pathology Department (Boston Children’s Hospital of Wisconsin, Milwaukee) following institutional review board approved protocol. These sections were cut at 4µm thickness, deparaffinized, and blocked with peroxidase block and serum-free protein block (Dako, Agilent Technologies). The slides were incubated with the primary, etv-2 (Abcam, Cambridge, United Kingdom #ab181847), c-Kit antibody (Dako), followed by secondary antibody. We tried using the DMAB-DCC056 antibody from Creative Diagnostics (New York, NY, USA) for GNAQ Q209L, but it did not work in our hands.

Serial sections of formalin-fixed, paraffin-embedded (FFPE) resected xenografts, 10-μm thick, were used for histopathology analyses by H&E staining. For all tumors, the histological diagnoses were confirmed under light microscopy by an experienced pathologist. Sections for immunohistochemical staining were treated twice with 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes and then in 0.3% hydrogen peroxide solution in order to block endogenous peroxidase activity. The sections were then blocked with serum followed by an Avidin-Biotin blocking reagent (Vector Laboratories; Burlingame, CA) in order to inhibit non-specific binding in the tissue. The sections were then incubated with polyclonal rabbit anti-human CD117 (c-KIT) antibody (1:50, Dako North America, Carpinteria, CA) overnight at 4°C. Sections were next incubated with biotinylated secondary antibody and ABC reagents of the Vectastain Elite Universal ABC kit according to the manufacturer’s instructions (Vector Laboratories). The secondary antibody was detected using the Avidin-Biotin-Peroxidase method with 3,3′-diaminobenzidine as the substrate (Vector Laboratories). Negative controls were performed by omitting the primary antibody and/or using isotype control antibody.