Horseradish peroxidase hrp linked anti mouse igg secondary antibody

We processed the Western blotting of retinas for the HIF-1α protein expression analysis. Fifty μg of protein per well was loaded on 8% SDS-polyacrylamide electrophoresis gel (SDS-PAGE) (P0012A, Beyotime, Beijing, China), followed by electro-transference to Polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A) for 2 hours at 200 mA. The membranes were blocked with 5% bovine serum albumin (BSA) (A3808, MultiSciences Biotech Co., Hangzhou, China) in tris-buffered saline with tween (TBST) for 2 hours and incubated with mouse monoclonal anti-HIF-1α antibody (1:1,500; Abcam, New Territories, Hong Kong) at 4°C overnight. After being washed with TBST, the membranes were incubated with a horseradish-peroxidase-(HRP)-linked anti-mouse IgG secondary antibody (1:6,000; Cell Signaling Technology, MA, U.S.A.) for 1 hour at room temperature and washed with TBST. β-actin (1:5,000; Cell Signaling Technology, MA, U.S.A.) was used as an internal control. The bands were detected by using a chemiluminescence kit (Millipore, Billerica, MA, U.S.A) and the bands' intensities were quantitatively analyzed using Image J 1.43 U software (Wayne Rasband, National Institute of Health, U.S.A.).