Rabbit anti col4a2

The samples were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin‐embedded tissues were dewaxed, followed by antigen retrieval. Sections were blocked by bovine serum albumin (BSA; Sinopharm Chemical Reagent) for 30 minutes after blocking endogenous peroxidase. Sections were then incubated with mouse anti‐vinculin (VCL, 1:200 dilution; MilliporeSigma, United States) and rabbit anti‐COL4A2 (1:200 dilution; Bioss, China) overnight. The corresponding secondary antibody (Dako, Denmark) was added to the tissues and incubated for 50 minutes at room temperature. Freshly prepared DAB coloring solution (Dako) was then added, and the color development time was determined after observation under a microscope. Excessive color was washed with tap water to terminate the color development. Finally, nuclei were counterstained, and the sections were dehydrated and sealed.

Proteins were lysed from rat aortic SMCs, and equal amount of protein per lane (20 μg) was separated by 12.5% SDS‐PAGE gel (Epizyme, China). Proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (MilliporeSigma). The membrane was blocked with a QuickBlock blocking buffer (Beyotime Biotechnology) for 15 minutes at room temperature, followed by incubation with different primary antibodies, including rabbit anti‐MYLK (1:5000 dilution; Abcam), rabbit anti‐COL4A2 (1:500 dilution; Bioss, China), rabbit anti‐SMA (1:100 dilution; Abcam), goat anti‐ transgelin (TAGLN, 1:500 dilution; Abcam), and mouse anti‐VCL (1:1000 dilution; Millipore) overnight. The membrane was then incubated with horseradish peroxidase‐conjugated secondary antibody for 1 h at room temperature. Immunoblots were probed using enhanced ECL substrate (Thermo). The blot was detected using an imaging system (Bio‐Rad), and the chemiluminescence level was recorded. The results were normalized to GAPDH. The experiments were replicated for three times.