Immunostaining Analysis of Vascular Cells

The hall makers of VSMCs and macrophages were identified by immunofluorescent stains which were performed in accordance with our previous descriptions [16 (link)]. After animals were sacrificed by CO₂ suffocation, aortic roots were dissected and fixed with paraformaldehyde (4%, v/w). 5 μm paraffin tissue sections were made. For H&E stain, sections were washed by PBS and then processed with an H&E stain kit (Beyotime). The vessel lumen area (A1) and internal elastic lamina area (A2) were measured with ImageJ software (version 1.53e, NIH). The stenosis percentage was calculated as follows: (A2 − A1)/A2∗100%. Fixed cells or slides were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). After blocking buffer (Abcam) incubation, slides were washed and treated with specific primary antibodies against inducible nitric oxide synthase (iNOS, 1 : 200, Abcam), myosin heavy polypeptide 11 (MYH11, 1 : 200, Abcam), RAGE (1 : 200, Abcam), and TLR4 (1 : 200, Abcam) at 4°C for 8 h. Slides were washed by PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 (Abcam) at 25°C for 20 min. Cell nuclei were stained with DAPI (Abcam). A fluorescence microscope (Nikon) was used to observe. The captured images were further analyzed by ImageJ software.