Vascular tissues were collected and fixed overnight in 4% paraformaldehyde (Solarbio, China), dehydrated, and embedded in paraffin. Sections were cut to a thickness of 4 μm, and an HE automatic staining instrument (HistoCore SPECTRA ST, Leica, Germany) was used to perform the HE staining. Images were taken using a digital slice scanner (KFBIO, China). The degree of stenosis and the neointima/media ratio were calculated as previously described (36 (link)).
For tissue immunofluorescence staining, paraffin sections were dewaxed in xylene and antigen-retrieved in citrate (Solarbio). Sections were blocked in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime) for 1 h at room temperature and incubated with anti-α-SMA (1:200; Proteintech, Cat# 67735-1-Ig) and anti-PCNA (1:100; Proteintech, Cat# 60097-1-Ig) primary antibodies overnight at 4°C. Fluorescent secondary antibodies Alexa Flour 594-conjugated goat anti-rabbit IgG (1:200, Proteintech, Cat# SA00013-4) and Alexa Flour 488-conjugated goat anti-mouse (1:200, Proteintech, Cat# SA00013-1) were used to create fluorescent signals. Sections were observed with a laser confocal microscope (Nikon, AXR, Japan).
For tissue immunofluorescence staining, paraffin sections were dewaxed in xylene and antigen-retrieved in citrate (Solarbio). Sections were blocked in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime) for 1 h at room temperature and incubated with anti-α-SMA (1:200; Proteintech, Cat# 67735-1-Ig) and anti-PCNA (1:100; Proteintech, Cat# 60097-1-Ig) primary antibodies overnight at 4°C. Fluorescent secondary antibodies Alexa Flour 594-conjugated goat anti-rabbit IgG (1:200, Proteintech, Cat# SA00013-4) and Alexa Flour 488-conjugated goat anti-mouse (1:200, Proteintech, Cat# SA00013-1) were used to create fluorescent signals. Sections were observed with a laser confocal microscope (Nikon, AXR, Japan).