Ab8226

Tissue slides were subjected to antigen retrieval with Tris buffer pH = 8.0 (Vector Labs H-3301) in a pressure cooker. They were then blocked in 5% normal donkey serum in phosphate buffered saline (PBS) containing 0.1% Triton-X, in combination with the avidin/biotin blocking reagent (Vector Labs SP-2001). Sections were incubated with primary and secondary antibodies and mounted in Prolong Gold with DAPI medium (Invitrogen). Biotinylated goat antibody (Jackson Immunoresearch 705-065-147) was applied to sections stained with TBX3, before detection with Streptavidin-647. GS and β-ACTIN staining was performed with the Mouse-on-Mouse detection kit (Vector Labs) according to the manufacturer’s protocol. The following antibodies were used: GFP (chicken, 1:500; Abcam ab13970), TBX3 (goat, 1:50; Santa Cruz sc-17871), GS (mouse, 1:500; Millipore MAB302), β-ACTIN (mouse, 1:100; Abcam ab8226), and HNF4α (rabbit 1:50; Santa Cruz sc8987). Samples were imaged at 20× magnification using a Zeiss Imager Z.2 and processed and analyzed with ImageJ software. For immunocytochemistry, plated cells were fixed with 4% paraformaldehyde, blocked in 5% normal donkey serum in PBS containing 0.1% Triton-X, and stained with primary and secondary antibodies as indicated above. Cells were imaged using a Zeiss Spinning Disk Confocal Microscope.

30–40 µg protein was resolved on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with primary antibodies against HIF-1α (BD Biosciences 610959, 1:1000), β-actin (Abcam ab8226, 1:1000) and p21 (Santa Cruz sc-396, 1:500) at 4 °C overnight. Membranes were incubated with either an anti-mouse (1:5000) or anti-rabbit (1:3000) horseradish peroxidase-linked secondary antibody (Cell Signalling) for 1 h at RT. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used according to the manufacturer’s instructions prior to detection using a G:BOX gel imaging system (Syngene, UK).