Axio vision imaging software v 4

Paraffin-embedded pancreatic sections were incubated with primary antibodies followed by visualization using fluorescent secondary antibodies as previously published [16 (link)]. Primary antibodies: rabbit anti-insulin at 1:500 (Abcam, Cambridge, MA) and rat anti-bromodeoxyuridine (BrdU) at 1:1000 (Abcam). Secondary Abs: Cy-2 coupled donkey anti-rat IgG antibody at 1:200 (Jackson Immuno Research, West Grove, PA); Cy-3 coupled donkey anti-rabbit IgG antibody at 1:200 (Jackson Immuno Research). Nuclei were visualized with 1 μg/ml 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA). Images of pancreatic sections were recorded with Axio Observer Z.1 fluorescent microscope with Axio Vision Imaging Software v 4.7.1.0 (Carl Zeiss, Oberkochen, Germany). Total beta cell area was measured via summation of insulin positive area in a pancreatic section with maximum footprint for each mouse using Mosai X (scan mode: meander, autofocus per tile) in Axio Vision. BrdU positive nuclei were manually selected if 1) it was surrounded by insulin and 2) superimposed with DAPI to pick up a proliferating beta-cell. The number of BrdU positive nuclei was corrected for total beta cell area of each mouse.