Western blotting analysis using anti-HIF1 alpha antibody (Cat#51123). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with anti-HIF1 alpha antibody (Cat#51123, 1:2,500) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).

Immunocytochemical analysis of HIF1 alpha staining in LO2 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with the primary antibody in 3% BSA-PBS and incubated overnight at 4 °C in a hidified chamber. Cells were washed with PBST and incubated with a AF488-conjugated secondary antibody (green) in PBS at room temperature in the dark. Phalloidin - AF594 was used to stain Actin filaments (red). DAPI was used to stain the cell nuclei (blue).

Immunohistochemical analysis of HIF1 alpha staining in human brain formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). The section was then incubated with the antibody at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.