Rabbit anti brdu

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.