Hydrogen peroxide 3

After deparaffinization and rehydration, sections were heated to 95 °C for 15 min in Citrate Buffer, pH 6.0 (Invitrogen), for antigen retrieval. Hydrogen peroxide 3% (Fisher Scientific) was used to block endogenous peroxidase activity for 15 min at RT. Sections were preincubated with normal serum for 20 min at RT, then incubated in primary antibody TGF-β1 (Abcam ab66043, 1:200) at 4 °C overnight. After washing in PBST, sections were incubated in biotinylated secondary antibody (Vector Labs, 1:200) for 30 min at RT followed by ABC reagent (Vector labs) for 30 min. Color was developed using ImmPACT™ DAB (Vector Labs). The sections were then counterstained with hematoxylin (Fisher Scientific), dehydrated, and mounted using permanent mounting media (Fisher Scientific). Slides with no primary antibody applied were used as negative controls. The staining results were validated by a blind review performed by a pathologist with extensive experience examining vascular anomalies and immunohistochemistry. Microscopy was performed and images were taken with a Biotek Cytation5 Imaging Reader (Agilent Technologies, California, USA) and Olympus BX43 light microscope with Infinity 3S camera (Olympus, Pennsylvania, USA), respectively.