Rat anti insulin

Immunofluorescence was conducted as follows: slides were washed three times for 5 min each in a potassium-based phosphate-buffered saline (KPBS) and then incubated with primary antibodies diluted in donkey block (KPBS supplemented with 2% donkey serum and 0.4% Triton X-100) overnight at 4°C. Slides were then washed three more times in KPBS and incubated with secondary antibodies (Jackson ImmunoResearch) used at a 1:600 dilution in donkey block for 45 min at room temperature. Slides were washed three more times before counterstaining with DAPI where applicable and embedding in ProLong Gold Antifade (Thermo Fisher Scientific). Images were captured with a Nikon A1R+ confocal microscope or an automated Keyence microscope for the data presented in Fig. 1.
Primary antibodies include guinea pig anti-insulin (cat. no. A0564, 1:500; Dako), rat anti-insulin (MAB1417, 1:500; R&D Systems), guinea pig anti-Ucn3 (044, 1:1,000) and rabbit anti-Ucn3 (7218, 1:1,000) (gifts from Dr. Wylie Vale, Salk Institute for Biological Studies) (23 (link)), and rabbit anti-glucagon (2760S, 1:400; Cell Signaling Technology). Click-iT Plus EdU Cell Proliferation Kit (C10637; Thermo Fisher Scientific) was used for EdU detection in pancreatic tissue sections.