Quantification of proliferating cells and apoptotic cells in decellularized scaffolds

Explanted intestinal grafts were fixed in 4% PFA, embedded in OCT and sectioned. Sections were stained for cleaved caspase 3 (Cell Signaling, 9661S) and for Ki67 (Abcam, AB15580). First, the sections were blocked for 1 h in PBS with 10% donkey serum. Then, primary antibodies were incubated overnight at 4 °C in blocking solution with the addition of 0.5% Triton-X. Primary antibodies were washed 3 times with PBS before the secondary antibody was added. Secondary antibody for donkey anti-mouse or rabbit (Alexa Fluor 547 or 647; Life Tech) was used at a dilution of 1:500 in blocking solution with 0.5% Triton X-100 and incubated at room temperature for 1 hour. Secondary antibody buffer was washed off with PBS 3 times and the slides mounted in a solution containing DAPI. Images were acquired with a confocal microscope (Zeiss LSM710). Three fields of view (425.10 μm × 425.10 μm in size) were evaluated per animal and the ratio between human VEcad (injected intra-vitally before euthanasia) and cleaved caspase 3- or Ki67-positive cells quantified.