Avidin and biotin block

Staining was performed on formalin fixed, paraffin embedded mouse pancreas cut at four microns and mounted on charged glass slides. Staining was done online using the Dako Autostainer (Dako Inc). The slides were pre-treated by a heat induced epitope retrieval method using an antigen retrieval buffer (Dako), followed by an avidin and biotin block (Vector Labs), and background block using Peroxidased 1 and Background Sniper (Biocare Medical) prior to antibody incubation. Slides were first stained for BrdU (Accurate Chemical), followed by a goat-anti-rat secondary step, followed by streptavidin-HRP, followed by a DAB chromogen (Dako) for detection of proliferating cells. Next, slides were stained for insulin (Dako), followed by a goat-anti-guinea pig secondary step, followed by an alkaline phosphatase step, followed by Vulcan Fast Red chromagen (Biocare Medical) for detection of beta-cells. All slides were counterstained with Modified Mayer’s Hematoxylin (DAKO). Morphometric analysis was performed using a Scan Scope XT (Aperio) and both Image Scope (Aperio) and Indica Lab (Indica Lab) software. For each animal, 5 sections, ≥ 300 microns apart, were evaluated.

All mice were sacrificed after 14 days. Their wounds were photographed, and the wound areas were excised, fixed in 4% formalin, embedded in paraffin, and sectioned at 100–200 μm intervals. Five serial sections (5 μm) were collected up to the maximal wound area to perform H&E staining. Sections of interest were further processed for immunohistochemistry. Antigen retrieval was performed at pH 6. Rat-anti-mouse CD31 (2 μg/mL, 1 h, Dianova) was used to identify murine blood vessels. Staining was visualized using a biotinylated rabbit-anti-rat antibody (10 μg/mL, VectorLab) and streptavidin-peroxidase reagent (10 min; ThermoScientific) after blocking with H2O2 (10 min), avidin and biotin block (both 15 min; VectorLab), and protein block (10 min). To detect hAMSCs, we performed antigen retrieval at pH 9. After blocking in H2O2 (10 min; ThermoScientific), UV block (5 min; ThermoScientific), M.O.M™ (1 h; VectorLab), and protein block (30 min; Dako), slides were exposed to anti-vimentin for 30 min (0.078 μg/ml; Dako) and developed using the UltraVision LP Detection System (ThermoScientific). Alternatively, we used anti Ki-67 (1.1 μg/ml; Dako) to detect proliferating hAMSCs and goat-anti mouse fluorescein isothiocyanate (BD) as a secondary antibody.