Anti cd31 pecam 1

Angiogenesis was evaluated on paraffin-embedded tissue sections by immunohistochemistry, assessing the micro-vessel density (MVD) and the endothelial cell VEGF expression according to Simonetti et al. [50 (link)]. The sections were incubated overnight at 4 °C with the anti-VEGF-C-1 (diluted 1:200, Santa Cruz, CA, USA) and anti-CD31/PECAM-1 (diluted 1:20, Santa Cruz, CA, USA) antibodies. The number of CD31-positive small vessels (MVD) was counted under a light microscope at a magnification of 400× covering an area of 0.16 mm² per field. We considered positive any brown stained endothelial cell or endothelial cell cluster clearly separated from adjacent micro-vessels. MVD was represented as number of counted micro-vessels per mm² while positive VEGF cells were expressed as percentage over total cells counted in the selected fields. All counts were performed by one investigator three times for each sample and expressed as the obtained mean values.

The reagents included carbon tetrachloride (Sigma‐Aldrich, 56‐23‐5), 30% H₂O₂ (Hydrogen peroxide 30%, Sigma‐Aldrich, 1.07298), selisistat (EX‐527, MedChemExpress, 49843‐98‐3), resveratrol (SRT501, MedChemExpress, 501‐36‐0), DAPI (Sigma‐Aldrich, D9542), Alexa Fluor™ 647 Phalloidin (Thermo, A22287), protease cocktails inhibitor (Beyotime, P1005) and PMSF (Phenylmethanesulphonyl fluoride, Beyotime, ST506).
The primary antibodies included anti‐α‐SMA (Boster, BM0002), anti‐vWF (Santa Cruz, SC‐365712), anti‐CD32b (ZEN‐bioscience, 382560), anti‐CD31 (PECAM‐1, Santa Cruz, sc‐18916), anti‐CD31 (Abcam, ab33858), anti‐NOX2 (Proteintech, 19013‐1‐AP), anti‐NOX4 (Proteintech, 14347‐1‐AP), anti‐Lamin A/C (Cell Signaling Technology, 4777S), anti‐Lamin B1 (Proteintech, 66095‐1‐Ig), anti‐progerin (Santa Cruz, sc‐81611), anti‐p53 (Abcam, ab131442), anti‐p53 (acetyl K381; Abcam, ab61241), anti‐SIRT1 (Abcam, ab110304), anti‐Histone H3 (Proteintech, 17168‐1‐AP) and anti‐GAPDH (Proteintech, 60004‐1). HRP‐conjugated Affinipure Goat Anti‐Mouse IgG (H + L; Proteintech, SA00001‐1), HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG (H + L; Proteintech, SA00001‐2), FITC‐labelled goat anti‐rabbit IgG (H + L; Beyotime, a0562) and Cy3‐labelled goat anti‐mouse IgG (H + L; Beyotime, a0521) were used for secondary antibodies.