The pancreas were fixed in 4% paraformaldehyde at 4°C overnight. The tissues were dehydrated, embedded in paraffin, and sectioned at 5 μm thickness. The primary antibodies used were as follows: rabbit anti-BrdU (1:50, Sigma-Aldrich), guinea pig anti-insulin (1:800, Dako), mouse anti-SFRP5 (1:50, Santa Cruz Biotech), and rabbit anti-glucagon (1:100, Santa Cruz Biotech). Incubations were performed overnight in a humidified chamber at 4°C. For BrdU/insulin staining, sections were treated with 1 M HCl at 37°C for 60 min before incubation with the primary antibody. Secondary antibodies were used as follows: Alexa Fluor 488 donkey anti-guinea pig (1:500, Jackson Immunoresearch), Alexa Fluor 488 donkey anti-rabbit (1:500, Jackson Immunoresearch), and Alexa Fluor 594 donkey anti-mouse (1:500, Life Technologies). Images were captured using an Olympus Microscope system.
After immunelabeling with BrdU and insulin, pancreatic islets in paraffin-embedded sections were photographed at ×400 and assigned blinded filenames. The number of BrdU (+) β-cells was manually counted. At least 2000 β-cells were counted per animal using three sections that were separated by at least 100 μm. Three animals per group were analyzed.
After immunelabeling with BrdU and insulin, pancreatic islets in paraffin-embedded sections were photographed at ×400 and assigned blinded filenames. The number of BrdU (+) β-cells was manually counted. At least 2000 β-cells were counted per animal using three sections that were separated by at least 100 μm. Three animals per group were analyzed.