Anti 11β hsd1 antibody

Treated DPCs were dissolved in PRO-PREP™ protein extraction solution (iNtRON Biotech, Seongnam, Korea). Total cell lysates (30 µg) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using Towbin buffer. Membranes were then blocked with 5% milk in TBS + 0.1% Tween-20 (Sigma Aldrich, Dorset, UK) and incubated with anti-11β-HSD1 antibody (1:500 dilution, Abcam), anti-vascular endothelial growth factor (VEGF) antibody (1:500 dilution, Abcam), anti-Wnt5a antibody (1:500 dilution, Abcam), or anti-alkaline phosphatase (ALP) antibody (1:500 dilution, Abcam) as primary antibodies and monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000, Beyotime Institute of Biotechnology, Nanjing, China) as a control. Blots were reacted with Immobilon Western reagent (Millipore) and detected using an Amersham Hyperfilm electrochemiluminescence (ECL) assay (GE Healthcare, Buckinghamshire, UK). Signals were detected using the ECL Plus Western blotting detection system (Amersham, Buckinghamshire, UK).

A total of 12 paraffin-embedded human scalp skin samples were obtained from patients undergoing excision for benign tumors of the scalp. For immunohistochemical staining, primary rabbit polyclonal anti-11β-HSD1 antibody (1:100 dilution, Abcam, Cambridge, UK) was reacted overnight at 4℃, and a horseradish peroxidase-conjugated secondary antibody was added to sections for 1 hour at room temperature. All sections were lightly counterstained with hematoxylin. This study was approved by the Institutional Review Board of Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, and all skin samples were obtained after receiving written informed consent from the donors (IRB No. 3-2015-0034).