Mouse anti gapdh igg

Western blot analyses were performed as previously described (Feng et al, 2011 (link)). The primary antibodies used were rabbit anti-hemagglutinin (HA) IgG, rabbit anti-Egr-1 (15F7) IgG (Cell Signaling Technology), mouse anti-GAPDH IgG, mouse anti-tubulin IgG, mouse anti-HuR (3A2) IgG, and goat anti-TIA-1 IgG (Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology).

Contralateral cortical tissue was collected 14 days after MCAO/r. The whole contralateral cerebral cortex was used for protein extraction, and the subcortical part was discarded. Whole protein extraction of the tissue samples was performed with RIPA lysis buffer (Beyotime, China) containing a protease inhibitor (Beyotime, China). Equal amounts of total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany). The membranes were blocked with 5% bovine serum albumin for 1 h and incubated with goat anti-CD31 IgG (1 : 1000, Santa Cruz Biotechnology, USA), rabbit anti-VEGF IgG (1 : 1000, Abcam, USA), mouse anti-eNOS IgG (1 : 3000, Sigma-Aldrich, USA), and mouse anti-GAPDH IgG (1 : 5000, Santa Cruz Biotechnology, USA) primary antibodies at 4°C overnight. After being washed with TBST, the PVDF membranes were incubated for 2 h at room temperature with the corresponding horseradish peroxidase- (HRP-) conjugated antibodies (1 : 5000, KPL, USA). The chemiluminescent signals were scanned by a ChemiDoc MP Imaging System with Image Lab Software (Bio-Rad, USA) and analysed with a Gel-Pro Analyser (Media Cybernetics Inc., Silver Spring, MD, USA).