Rabbit anti cleaved caspase 3

Cryo-sections were blocked for two hours with 10% horse serum in 0.1% Triton-X in 1XPBS followed by primary antibody staining with mouse anti-α-smooth muscle actin (Chemicon International) at a concentration of 1:100, rabbit anti-Ki67 (abcam) at a concentration of 1:500, or rabbit anti-cleaved caspase3 (R&D Systems) at a concentration of 1:1000 overnight at 4°C. Secondary antibodies conjugated with either donkey anti-mouse Alexa Fluor 488 or donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific) were used at a concentration of 1:1000 for two hours at room temperature. Three 15-minute 1XPBS washes were performed between antibody incubations. Fluorescent images were visualized and photographed on a Zeiss M1 Epifluorescent microscope with Axiovision software.

Histopathology and immunohistochemistry were performed on frozen tissue sections. Frozen sections were air dried and fixed with 3% formaldehyde. Hydrogen peroxide was used to quench endogenous peroxidases. Tissue was blocked with 10% normal goat serum in PBS with 0.1% Tween-20. Primary antibodies against mouse antigens included: rat anti-F4/80 (eBioscience, San Diego, CA), rat anti-Ly6C (Abcam), rat anti-Ki67 clone TEC3 (Dako), rat anti-CD31 (BD Pharmingen), rat anti-CD45 (BD Pharmingen) and rabbit anti-cleaved caspase-3 (R&D Systems). Tissues were incubated in blocking buffer containing primary antibodies overnight at 4ºC. After washing with PBS, tissues were incubated with PBS containing a secondary biotinylated goat anti-rat antibody (BD Pharmingen). Tissues were developed using Vectastain ABC kit and counterstained with hematoxylin. Senescence associated Beta-galactosidase staining was performed using the Senescence β-Galactosidase Staining kit (Cell Signaling Technology). Fluorescent imaging was performed on an IX83 inverted microscope (Olympus) and brightfield images were acquired using a BX43 upright (Olympus) microscope.