Tcs sc8

Surgically removed Panc02 tumors were embedded and frozen in OCT compound. Cryostat sections (8-μm thick) were fixed in 4% paraformaldehyde for 10 min and blocked with 1% BSA in DPBS. The sections were incubated with rat anti-mouse monoclonal CD31 antibody (1:100 dilution) (BD Biosciences, Franklin Lake, NJ, USA) followed by incubation with Alexa Fluor 594-conjugated goat anti-rat IgG (1:300 dilution) (Thermo Fisher Scientific) and counterstaining with DAPI. Images were taken with a confocal laser scanning microscope (TCS SC8, Leica Microsystems, Wetzlar, Germany), and pixel values of the CD31-positive areas were calculated for each image to determine the tumor vessel density using ImageJ software (National Institutes of Health).

Tumour tissues were fixed in 4% paraformaldehyde solution. The tissues were embedded in paraffin and cut into 2- or 6-µm sections. After heat-induced antigen retrieval in REAL Target Retrieval Solution (DAKO) and subsequent blocking of nonspecific sites with 0.1% normal goat serum/1% BSA, the tissues were immunostained with polyclonal anti-Ki67 antibody (Novus Biologicals, Littleton, CO, USA) and polyclonal anti-cleaved caspase-3 (Asp175) antibody (CST) diluted 1:200 followed by Alexa Fluor 594-conjugated goat anti-rat IgG staining. Sections were counterstained with DAPI and observed under a confocal laser scanning microscope (TCS SC8, Leica Microsystems, Wetzlar, Germany).