Mea53200

Pancreas of mice were weighed, fixed with 4% paraformaldehyde and then embedded in paraffin to prepare 4 μm pancreatic sections. In brief, the paraffin sections of mouse pancreases were deparaffinized, rehydrated, antigen unmasking (Vector Laboratories, Inc., United States), blocking, and incubated overnight at 4°C with anti-insulin antibody (1:200 dilution) (ab7842, Abcam). In the next day, the sections were washed and stained by FITC secondary antibody. Then the β-cell areas and the entire tissue areas were scanned by a Nikon MEA53200 (Nikon, Japan) microscope. The cross-sectional areas of pancreas and β-cells were determined and calculated by NIS-Elements software (Nikon). The average β-cell mass of each mouse was calculated as follows: the islets/pancreas area ratio × the pancreatic weight. Five consecutive sections from each pancreas (6 mice per group) were used for β-cell mass measurements.

Cultured mouse islets in 6-well plates were fixed with 4% paraformaldehyde followed by permeabilization with 0.5% Triton X-100. The 4-μm paraffin sections of mouse pancreas were deparaffinized, rehydrated, and antigen unmasking. Then islets or sections were incubated with blocking buffer for 1 h at RT followed by incubating overnight at 4°C with antibody of insulin (Abcam), or Ki67 (1:50) (BD Pharmingen), or PDX1 (1:100) (Abcam). In the next day, islets or sections were washed by TBS and incubated by FITC- or Cy3-conjugated secondary antibodies (1:400) (Abcam) for 1 h at RT. β-cell apoptosis of pancreatic setions was detected by the commercial TUNEL staining kit according to the manufacturer’s instructions (TUNEL Brightred Apoptosis Detection Kit,Vazyme Biotech Co.). β-cell apoptosis of cultured islets was analyzed by the commercial TUNEL staining kit (In Situ Cell Death Detection Kit, TMR red; Roche Diagnostics). Slides were mounted with Vectashield Mounting Medium containing 4′6-diamidino-2-phenylindole (DAPI) as nuclear dye (Vector Laboratories, Inc., United States). Fluorescence was detected and analyzed using an Nikon MEA53200 (Nikon) microscope.

For morphometric data, ten sections (spanning the width of the pancreas) per mouse were analyzed. Pancreatic tissue area and insulin-positive area were determined by computer-assisted measurements by using a Nikon MEA53200 (Nikon GmbH, Dusseldorf, Germany) microscope, and images were acquired by using NIS-Elements software (Nikon). β-cell mass was obtained by multiplying the β-cell fraction by the weight of the pancreas.