Immunofluorescence Assay of Pancreatic Insulin

Briefly, 5 µm thick paraffin sections of pancreatic tissues were collected onto positively charged slides prior to staining with hematoxylin and eosin (H&E) for microscopic evaluation or immunofluorescence assay (IFA), as previously described [14 (link)]. For IFA, slides were rehydrated using previously outlined methods of toluene and ethanol changes [14 (link)] followed by antigen retrieval using Citrate Unmasking Solution (Cell Signal, Danvers, MA, USA). Slides were blocked with 10% goat serum for 1 h followed by overnight incubation with a rabbit polyclonal anti-insulin antibody (4590, Cell Signaling Technology, Danvers, MA, USA). Anti-rabbit Alexa Fluor 555 was used as the secondary antibody, as well as DAPI for a nuclear counterstain (Cell Signaling Technology, Danvers, MA, USA). All slides were assigned a quantitative histologic score based on relative total number of cells and fluorescent intensity by a board-certified veterinary pathologist blinded to study groups to ensure scientific rigor and reproducibility.