Ghs280

Similar to our previous report^{39 (link)}, mice were euthanized under anesthesia with CO₂ followed by cervical dislocation. The pancreas was immediately dissected, weighed, and fixed in 10% formalin on ice for 30 minutes. Pancreata were then washed in PBS and transferred through a series of solutions, beginning with 30% sucrose in PBS, 1:1 30% sucrose:OCT, and OCT before cryopreservation in OCT and storage at −80 °C. 10-micron sections, separated by 200 microns, were cut on positively charged slides. For each pancreas, one slide, containing 3 distinct sections, was post-fixed, quenched of peroxidase activity with 3% H₂O₂, and immunohistochemically labeled using guinea pig anti-insulin primary antibody (Dako A056401-2), diluted 1:500 in antibody diluent, and co-stained with hematoxylin (Sigma, GHS280). Slides were imaged using an automated pan-and-stich microscope at 10× (Evos). β-cell fractional area was determined by quantifying the percent of insulin-positive pancreas area as a total of the full pancreas area for each section, followed by averaging of 3 distinct sections per mouse. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) with shading correction. β-cell mass was calculated by multiplying β-cell fractional area by the pancreatic wet weight.