Mouse anti klf4 antibody

Murine and human aortas were fixed in 4% paraformaldehyde embedded in paraffin. Four ‐micrometers thick cross sections were prepared for hematoxylin and eosin stain and Elastica‐van Gieson staining for morphological assessment. For immunohistochemistry, rabbit anti‐VPO1 antibody VPO1 (5 μg/mL; EMD Millipore, USA), rabbit anti‐3‐Cl‐tyr antibody (2.5 μg/mL; Cell Science, USA), mouse anti‐KLF4 antibody (5 μg/mL; Abcam, UK), rabbit anti‐α−SMA and SM‐22α antibodies (1.25 μg/mL; Sigma, USA), and rabbit anti‐MMP‐2 antibody (2.5 μg/mL; Abcam, UK) were used. Paraffin sections were rehydrated and endogenous peroxidase activity was blocked for 30 minutes in methanol containing 0.3% hydrogen peroxide. Five percent normal goat serum (Sigma‐Aldrich, St. Louis, MO, USA) was incubated for 30 minutes at room temperature to block non‐specific background staining. Primary antibodies were incubated at 4°C overnight, followed by 60 minutes in biotinylated secondary antibody (2 μg/mL; Abcam, England). All specimens were counterstained with hematoxylin staining solution (Beyotime Institute of Biotechnology, China). Sections were scanned using OLYMPUS CX41 and Leica Application Suite 4.0 software. Morphological analysis and collateral degree was determined.

VSMCs were seeded on glass coverslips and fixed using 4% paraformaldehyde in PBS for 15 minutes at room temperature. Cells were then rinsed 3 times in PBS and subsequently blocked and permeabilized in 5% bovine serum albumin (Sigma‐Aldrich, St. Louis, MO, USA) dissolved in PBS containing 0.1% Triton X‐100 (Sigma‐Aldrich) for 120 minutes at room temperature. Cover slips were then rinsed and incubated with mouse anti‐KLF4 antibody (5 μg/mL; Abcam, UK), rabbit anti‐α‐SMA and SM‐22α antibodies (1.25 μg/mL; Sigma, USA) overnight at 4°C. Cover slips were then rinsed with PBS and incubated with antibodies labeled with Alexa Fluor dye with a maximum excitation at 488 nm (green; rabbit or mouse; 5 μg/mL; Abcam, UK) or for red with Alexa Fluor 594 nm (red; rabbit or mouse; 5 μg/mL; Abcam, UK) for 1 hour at 37°C in the dark. Finally, cover slips were incubated in DAPI (5 μg/mL; 4′, 6‐diamidino‐2‐phenylindole; Sigma‐Aldrich). Images were acquired using confocal microscopy (DM14000B, Leica, Germany).