Ab30788

For animals used to confirm the location of the preBötC with respect to labeling, animals were deeply anesthetized with 30 mg/kg tiletamine-zolazepam/10 mg/kg xylazine and transcardially perfused with ice-cold PBS followed by ice-cold 1% formalin PBS. Brains were then extracted and placed into 4% formalin PBS overnight at 4°C. Brains were placed into PBS for 2 days, then sliced in 100 μm thick slices using a Compresstome. Free-floating slices were then permeablized with 0.25% Triton X-100 PBS for 10 min, followed by a 30 min block with 1% bovine serum albumin in 0.1% Tween-20 in PBS (BSA-PBST). Slices were then incubated for 4 days at 4°C in BSA-PBST containing the antibody for somatostatin (1:100, ab30788, Abcam, Cambridge, United Kingdom). After washing with PBST, slices were incubated with a secondary antibody (anti-Rat, 1:400, Jackson ImmunoResearch Europe, Suffolk, United Kingdom) at room temperature for 1 h. Slices were washed and mounted with Permount (Fisher Scientific, Reinach, Switzerland), and imaged on an inverted fluorescence microscope (Nikon).

Pancreas samples were harvested from Hadh^lox/lox, SCHADKO, and β-SKO mice and immediately fixed in 4% paraformaldehyde solution overnight at 4 °C. Samples were then stored in PBS at 4 °C until processing and paraffin embedding. Immunostaining (5-μm tissue sections) for SCHAD and insulin was performed as previously described (14 (link)). For glucagon and SST staining, the sections were incubated overnight at 4 °C with mouse monoclonal anti-glucagon antibody (Sigma-Aldrich, G2654; 1:8000) or rat monoclonal anti-SST antibody (Abcam, ab30788; 1:500), followed by incubation for 1 h at room temperature with secondary antibody 1:400 from Jackson ImmunoResearch Laboratories (glucagon: #715-586-151; SST: #712-586-153).