Anti pcna

Cryosections with a thickness of 4 μm were prepared using a cryostat and were fixed in 4% paraformaldehyde for 15 min. After blocking, the cryosections were incubated with primary antibodies and then with a fluorescein Cy3-FITC-labelled secondary antibody (1:100; Proteintech, Wuhan, China). Fluorescence images were recorded using a TCS SP5II confocal microscope (Leica, Bensheim, Germany). The following primary antibodies were used: anti-desmin (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-podocalyxin (1:100; R&D Systems, Minneapolis, MN, USA), and anti-snail2 (1:100, Proteintech). Podocytes were seeded onto clean glass coverslips, fixed with 4% paraformaldehyde, and permeabilised with 0.2% Triton X-100. The slides were incubated with an anti-PCNA (1:100, Proteintech), anti-synaptopodin (1:100; Proteintech), anti-CDK4 (1:100; Abcam, Cambridge, UK), anti-P-YAP (1:100; Cell Signaling Technology, Danfoss, MA, USA), or anti-snail2 (1:100, Proteintech) antibody.
For immunohistochemistry analysis, after deparaffinisation, rehydration, antigen retrieval, and blocking, the sections were incubated with an anti-PCNA (1:100), anti-CDK4 (1:100), anti-desmin (1:100), anti-YAP (1:100, Proteintech), or anti-cyclin D1 (1:100, Cell Signaling Technology) primary antibody and then with a horseradish peroxidase-labelled secondary antibody (Beyotime, Shanghai, China).

After fixing, embedding, sliding, and deparaffinizing, tissue sections were blocked with 3% H₂O₂ and 5% BSA. Then sections were incubated with anti-FOXD1(Invitrogen, Cat No. PA5-35145), anti-KI67 (Proteintech, Cat No. 27309-1-AP), anti-PCNA (Proteintech, Cat No. 10205-2-AP), and anti-GLUT1(abcam, Cat No. ab115730) antibodies at 4 °C overnight. After washing with PBS, immunohistochemical secondary antibody was applied to the sections for 1 h at room temperature, followed by DAB staining, hematoxylin re-staining, and imaging. The results were evaluated blindly by two independent pathologists.

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).