Pancreatic Beta Cell Quantification

In brief, and as described previously [12 (link)], parallel pancreatic tissue sections were stained using standard protocols, buffers and diluents for insulin (rabbit anti-mouse insulin polyclonal antibody; 4590, Cell Signaling Technology), followed by incubation with horseradish peroxidase (HRP)-labelled polymer-conjugated goat anti-rabbit IgG (Dako EnVision+ System) and counterstaining with haematoxylin. Beta cell area was quantified from the total area (insulin-positive cells compared with non-positive tissue) using ImageJ (v1.53i; https://imagej.nih.gov) on consecutive pancreatic serial sections cut at 200 µm intervals. Beta cell mass (per mg) was calculated by multiplying the relative insulin-positive area by the mass of the isolated pancreas before fixation. Images were captured using a Leica DM 4000 or Leica DM 6000 Power Mosaic microscope (Leica Microsystems).