Ecl antirabbit igg horseradish peroxidase linked whole antibody

Cells were washed with ice-cold PBS 3 times on ice, suspended with 100 μl of 1x NuPAGE LDS sample buffer (Invitrogen) containing NuPAGE Sample Reducing Agent (Invitrogen), and then the cell suspension was boiled at 80C for 10 min. After electrophoresis and transferring to iBlot 2 NC Regular Stacks (Invitorgen), membranes were blocked with 5% skim milk in TBS with 0.1% Tween 20 (Sigma) (TBST) at RT for 1h, and then incubated with primary antibodies (diluted with TBST containing 1% skim milk) at 4C overnight. The dilution ratio is as followed; rabbit anti-platelet-derived growth factor receptor (PDGFR)-α (1:1000, Sigma) for oligodendrocyte precursor cell (OPC), rat anti-GFAP (1:1000, Thermo Fisher) for astrocyte, mouse anti-β-actin (1:10000, Sigma-Aldrich) for loading control. After washing with TBST 5 times at RT, membranes were incubated with secondary antibodies (diluted with TBST containing 1% skim milk). The dilution ratio is as followed; ECL anti-rabbit IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare), anti-rat IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare), anti-mouse IgG, horseradish peroxidase linked whole antibody (1:1000, GE Healthcare). After washing with TBST 5 times at RT, membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific), according to the manufacturer’s protocol.