Immunoblotting
At POD7, the rats were sacrificed. From area II, six 0.5×0.5 cm samples
were collected for western blot analysis. To extract the flap tissue
protein, lysis buffer was utilized, and using the BCA assay,
concentration was determined. PAGE (12%) was used to separate the
protein, which was then transferred to the PVDF membrane. At room
temperature and after 2 hours and using 5% (w/v) nonfat milk, the
membrane was blocked, then underwent incubation with the primary
antibody VEGF (1: 1000), eNOS (1: 1000), MMP9 (1: 1000), HO1 (1: 1000),
cadherin 5 (1: 500), Bax (1: 500), SOD1 (1: 1000), CYC (1: 1000),
caspase 3 (CASP3) (1: 1000), and GAPDH (1: 1000)) at 4°C overnight, then
at room temperature, it underwent re-incubation with HRP-conjugated IgG
secondary antibody for two hours. To visualize the membrane, the ECL
reagent kit was employed. To assess the blots intensity, the Image Lab
3.0 software was utilized.