Quantifying Pancreatic Cell Populations

Isolated pancreata were fixed in 10% (v/v) formalin for 24 hours and embedded in paraffin wax. Sections (5 μm) were cut and fixed on Superfrost slides. Slides were permeabilized as detailed in Ref. 38 (link) and blotted with the following primary antibodies: antiguinea pig insulin (1:200; Dako) and antirabbit glucagon (1:100; Santa Cruz Biotechnology). Slides were visualized using an Axiovert 200M microscope (Zeiss) with Alexa Fluor 488 goat antiguinea pig IgG and with Alexa Fluor 568 donkey antirabbit IgG (Invitrogen). For examination of apoptosis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were performed on the above sections using a DeadEnd Fluorometric TUNEL system kit (Promega) according to the manufacturer's instructions. ImageJ software (Wayne Rasband, National Institute of Mental Health) was used to calculate the mean intensity of insulin staining in the insulin-positive area, β- and α-cell mass, and the number of TUNEL-positive cells of all visible islets. For β-cell mass estimation by immunocytochemistry, we determined the percentage of pancreatic surface that was insulin positive, as measured in whole pancreas sections separated by 25 μm in the z-axis.