Ab197493

As with immunohistochemistry staining, sections of HUVECs were incubated in a dark box with HIF primary antibody (ab197493, Abcam, Danvers, MA), and appropriate fluorescence-conjugated secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China). DAPI reagent was used to stain the cell nucleus. All of the images were obtained using fluorescence camera microscopy (Leica, Wetzlar, Germany). The results were based on the five areas of positivity observed, and the proportion of positive staining area was calculated by Image J IHC Profiler software.

Ischemic brain tissue (cell protein) extract was assayed with a BCA kit according to the manufacturer’s protocols towards obtaining accurate concentration of proteins and then added with 1/4 volume of protein buffer and denatured by heating. Aliquots of the samples (40 μg) were analysed through SDS-PAGE gel. Following this, the gel was transferred and then blocked and incubated with different corresponding primary antibodies against HIF-1α (ab197493, Abcam, Danvers, MA), VEGF (A12303, ABclonal, Wuhan, China), VEGFR2 (YT5845, Immunoway, Plano, TX), PLCγ1 (AF8390, Affinity, San Francisco, CA), PKC (YT3752, Immunoway, Plano, TX), eNOS (CPA9156, Bejing, China) overnight. Subsequently, the target membranes were incubated with a secondary antibody. Proteins were detected using the ECL kit and the results were analysed using Image Lab analysis software (Bio-Rad Laboratories, Inc., Hercules, CA).