Horseradish peroxidase hrp conjugated goat anti mouse igg h l

All rats fulfilling the inclusion criteria were decollated, and arterial occlusive tissues were snap frozen in liquid nitrogen and stored at −80°C for further use. Western blot analyses were performed to detected concentration of CaM, Gq proteins as described previously with some modifications [24 (link), 25 (link)]. Briefly, samples were homogenized in cold lysis buffer, and protein concentrations were determined using a BCA protein assay kit (Beyotime Corporation, China). Cell lysates were analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After transfer, membranes were blocked by incubating with 5% (w/v) nonfat dry milk in PBS solution with 0.05% Tween 20 for 1 h at room temperature or overnight at 4°C. Membranes were then incubated with the primary antibodies (anti-calmodulin antibody 1:1000 diluted and anti-GNAQ + GNA11 antibody 1:800 diluted), followed by secondary antibodies (horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG-H&L and HRP-conjugated goat anti-rabbit IgG-H&L), which were all purchased from Abcam (Cambridge, MA, USA), and developed using the enhanced chemiluminescence method (Pierce). Densitometry analysis was carried out using the Quantity One program (Biorad, Hercules, CA, USA).