Gb13097

Paraffin sections (LEICA company, Germany) were deparaffinized and rehydrated in graded alcohols. In addition, antigens were retrieved, autofluorescence quencher was added, and bovine serum albumin was added for incubation. Furthermore, primary antibodies anti-insulin mouse mAb (GB13121, Servicebio, China) and anti-glucagon rabbit pAb (GB13097, Servicebio, China) were added dropwise to the sections, incubated overnight, washed, incubated with corresponding secondary antibodies, and washed. Besides, DAPI (Solarbio, C0060) staining solution was added dropwise, and anti-fade mounting medium was adopted to mount the slides. Then, images were observed and collected under the fluorescence microscope (Leica DM2500 microscope: Leica, Germany).

Paraffin-embedded pancreatic sections were incubated with specific primary and secondary antibodies, then observed under Axiovision Observer A1 fluorescence microscope (Carl Zeiss, Germany) and photographed using the accompanying software. In accordance with former researches [38 (link), 41 (link)], quantifications of insulin, glucagon and F4/80 positive area proportions per islet were performed using the manual histology tools in Image J Software (NIH, Bethesda, MD, USA). Four pairs of 4-μm-thick serial sections, 100 μm apart were analyzed. An average of 53 islets from 3 to 4 mice for each group were analyzed for insulin and glucagon areas, and 67 islets from 6 mice per group were assessed for F4/80 areas.
Primary antibodies for Insulin (#GB13121), Glucagon (#GB13097), F4/80 (#GB11027) and Cy3/Alexa 488 conjugated fluorescent secondary antibodies (#GB21301/#GB25303) were all purchased from Servicebio Biotechnology (Wuhan, Hubei, China). Fluorescent stain solution 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for nuclei labeling was bought from the same company.

The pancreatic paraffin sections were dewaxed and dehydrated. The tissue slices were incubated overnight with the primary IR (Servicebio, GB11334, 1:300) and glucagon resistance (Servicebio, GB13097, 1:100) antibodies at 4 °C. The tissue slices were then washed and incubated at room temperature for 1 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Servicebio, GB25303, 1:400) and Alexa Fluor Cy3-conjugated goat anti-mouse IgG (Servicebio, GB21303, 1:300). The slices were rewashed and stained with DAPI for 10 min at room temperature. A confocal fluorescence microscope (Ti-U, NIKON, Japan) was used to obtain images of the slices.