Rat anti brdu

Mouse pancreata were analyzed by immunostaining using the following primary antibodies: guinea pig anti-insulin (Dako), 1:1000; rabbit anti-Ki67 (Thermo Fisher Scientific), 1:200; rat anti-BrdU (Novus Biologicals), 1:250; goat-anti-GFP (Abcam), 1:1000; rabbit-anti-MafA (Bethyl Labs), 1:1000; rabbit-anti-Pdx1 (Abcam), 1:500; rabbit-anti-Nkx6.1 (LifeSpan BioSciences), 1:250; mouse-anti-glucagon (Sigma), 1:100; mouse-anti-somatostatin (BCBC), 1:2000. Primary antibodies were detected with donkey-raised secondary antibodies conjugated to Cy3 or Cy5, (Jackson ImmunoResearch), and nuclei were counterstained with DAPI (Sigma) at 0.1 μg/ml. Images were captured on a Zeiss Axio Observer Z1 microscope with an ApoTome module and processed with Zeiss AxioVision 4.8 software. For beta-cell mass measurements, images covering an entire pancreas section were tiled using a Zeiss Axio Oberver Z1 microscope with the Zeiss ApoTome module. The insulin+ and total pancreas areas were measured using ImageJ and beta-cell mass was calculated as follows: Insulin+ area/total pancreatic area. For examination of apoptosis, TUNEL analysis was performed using ApopTag Red In Situ Apoptosis Kit as specified by the manufacturer (Thermo Fisher Scientific).