Anti cd31 rabbit polyclonal antibody

Under normal conditions, proteins in the lysates of the cells treated with CoCl₂ and/or oligomycin A were subjected to 8.5 or 12.5% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes (Immobilon-FL, Merck-Millipore, Cork, Ireland) . The membrane was blocked with 5% skim milk (Snow Brand, Japan) and incubated with the primary antibodies overnight at 4 °C followed by incubation with the suitable secondary antibody. The primary antibodies used were anti-Oct-4A (9B7) mouse monoclonal IgG (1: 1000, #4286, Cell Signaling Technology, MA), anti-HIF-1α mouse monoclonal IgG1 clone # 241,809 (1: 1000, R&D Systems, MN), anti-CD31 rabbit polyclonal antibody (1:500, ab28364, Abcam, UK), anti-mouse β-tubulin rabbit polyclonal antibody (1: 1000, #2146 s, Cell Signaling Technology), anti β-actin mouse monoclonal antibody (1:2000, 010–27,841, Wako) and anti-GFP goat polyclonal antibody labeled with HRP (1: 2000, ab6663, Abcam, UK). The secondary antibodies used were anti-rabbit IgG goat IgG linked with HRP (1:10,000, #7074 s, Cell Signaling Technology)and anti-mouse IgG goat IgG linked with HRP (1: 10,000, #7076 s, Cell Signaling Technology). HRP reaction was performed with Ez West Lumi plus (ATTO, Japan), and the developed fluorescence intensity was detected by Light Capture II (ATTO, Japan) or Lumino Graph I (ATTO, Japan).

The CAM with the transplanted graft was embedded in paraffin blocks and cut into 6-μm-thick sections. The sections were deparaffinized and stained with hematoxylin and eosin (HE). For immunohistochemistry, the sections were stained with anti-Desmin mouse monoclonal antibody (clone: ab8470, dilution 1:50, Abcam, Cambridge, MA, USA), anti-CD31 rabbit polyclonal antibody (clone: ab28364, dilution 1:80, Abcam, Cambridge, MA, USA), and anti-ASCT2 rabbit polyclonal antibody (clone: HPA035240, dilution 1:50, Sigma-Aldrich, St Louis, MO, USA).