Postnatal Pancreas Development Analysis

P1 pups were intraperitoneally injected with BrdU (100 mg/kg; Sigma-Aldrich). Their pancreata were fixed in 4% PFA in PBS for 12 h at 4 °C followed by dehydration, paraffin embedding, and histologically analyzed as described previously (Lee et al, 2007 (link); Wei et al, 2014 (link)). For β-cell proliferation analysis in P1 pups every other section was labeled with guinea pig anti-insulin (1:200; DAKO A0564) and mouse anti-BrdU (1:500 Sigma B2531) antibodies, followed by fluorochrome-coupled secondary antibodies (Millipore) and DAPI counterstaining (Abcam). β-cell proliferation was quantified by counting the number of BrdU/insulin-positive cells over the total number of insulin-positive cells using ImageJ software. An average of 2000 insulin-positive cells per specimen was counted. Bmal1 and Clock levels were assessed on pancreas sections of P1 pups following co-labeling with rabbit anti-Bmal1 (1:500 Abcam ab3350) or rabbit anti-Clock (1:1000 Abcam ab3517) antibodies and a guinea Pig anti-insulin (1:800 DAKO A0564) antibody while Pck1 levels were assessed using a rabbit anti-PCK1 (1:400 Abcam ab70358) antibody, followed by fluorochrome-coupled secondary antibodies (Invitrogen A21206 and A21450).