Pancreatic Tissue Analysis and Staining

Pancreatic tissue was prepared and stained as previously described^{14 (link)}. Primary antibodies were ARC (Cayman Chemical), insulin (Abcam), BrdU (Roche), cleaved caspase-3 (Cell Signaling Technology), and α-tubulin (Sigma-Aldrich). Alexa Fluor 488 and 568 (Invitrogen) of corresponding species were used for recognition of primary antibodies. All images were collected with Axio Observer.Z1 microscope (Zeiss). Islet morphology was designated as abnormal if >25% of perimeter was jagged on hematoxylin and eosin-stained sections. All islets (~85–100) in pancreatic section were scored. β-cell area was determined by calculating the number of β-cell nuclei (insulin-positive cells) in an islet divided by the respective islet area (quantified with ImageJ; National Institutes of Health). A minimum of five islets were scored per mouse. For BrdU staining, BrdU 2.2 mg/ml was included in the drinking water for 4 days prior to sacrifice, and tissue sections were treated with 2 M HCl for 10 minutes at room temperature immediately following antigen retrieval. TUNEL staining was performed as previously described^{14 (link)}. Slides were coverslipped with VECTASHIELD mounting media with DAPI (Vector Laboratories).