Anti brdu

For immunohistology, mouse tissues were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin as described previously[60 (link)–62 (link)], then followed by hematoxylin and eosin staining (H&E staining) according to standard procedures. Afterward, the sections or slides were stained with immunohistochemistry (IHC). The primary antibodies are anti-insulin [1:100, Cell Signaling Technology (CST), USA], anti-glucagon (1:100; CST, USA), anti-Ki67 (1:300; Abcam) and anti-BrdU (1:50; GE Healthcare).

BrdU (bromodeoxyuridine) assay was carried out to measure the amount of proliferating cells wherein cells were incubated with BrdU (Sigma, St. Louis, MO) solution for 1 h at 37°C. Cells were washed with PBS and fixed in 4% para-formaldehyde. Permeabilization in PBS containing 0.1% triton was followed by incubation with 1 (N) HCl on ice for 10 mins and 2 (N) HCl at RT for 10 mins. Phosphate citric acid buffer pH 7.4 was added and cells were incubated at RT for 10 mins prior to washes with PBS containing 0.1% triton. Cells were blocked in 5% bovine serum albumin for 1 h at 4°C and incubated overnight with anti-BrdU (1:500) (Cat #RPN202, RRID : AB_2314032, GE Healthcare, Buckinghamshire, UK) at 4°C. Following day, cells were incubated with Alexa Fluor 594-conjugated anti-mouse secondary antibody (Cat #A-11032, RRID : AB_2534091, 1:1000) (Invitrogen, Carlsbad, CA) for 1 h at RT. Cells were washed with PBS and nuclei were counterstained with DAPI. Cells were mounted on slides and observed under fluorescence microscope (Nikon-Eclipse-Ti-S, Tokyo, Japan). The number of BrdU positive cells was counted and graphically presented relative to the control.