Immunohistochemistry of Pancreatic Tissues

Paraffin sections for pancreatic head and tail were prepared as previously described [30 (link),31 (link)]. The entire pancreas was sectioned every 200 μm, generating 8-16 sections for both head and tail pancreas. Primary antisera included guinea pig anti-insulin (catalogue number A0564, Dako, Carpinteria, CA, USA) and mouse anti-human Ki67 (catalogue number 550609; BD Biosciences, San Jose, CA, USA), followed by secondary antisera conjugated to Cy3 or Cy5 (catalogue numbers 706-166-148 and 715-605-151; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and DAPI (Molecular Probes, Eugene, OR, USA). EdU was detected by Click-iT EDU Alexa Fluor 647 Imaging kit (Invitrogen, Carlsbad, CA, USA). Images were acquired using Zeiss AxioImager (Carl Zeiss MicroImaging, Thornwood, NY, USA) with Orca-ER digital camera (Hamamatsu, Middlesex, NJ, USA). Slides were imaged to quantify beta cell morphometry as previously described [30 (link)], using Volocity 6.1.1 software (PerkinElmer, Waltham, MA, USA).