Mouse anti glucagon antibody

Pancreas was harvested from male mice, postfixed in 4% paraformaldehyde, rinsed in 70% ethanol, and embedded in paraffin. Paraffin sections (4 μm) were prepared. Pancreas sections were immunostained for insulin (guinea pig anti-insulin antibody, 1 : 800, Sigma, USA) and glucagon (mouse anti-glucagon antibody, 1 : 1000, Abcam, ab92517) and counterstained with DAPI (Olympus, Tokyo, Japan) to identify nuclei.

A portion of the pancreatic tail was fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained with Hematoxylin and Eosin (H&E). For fluorescent microscopy imaging of insulin and glucagon, paraffin sections (2 μm) were put onto microscope slides, deparaffinized in xylene, and hydrated through graded ethanol to distilled water. Tissue sections were permeabilized in PBS with 0.1% Triton X-100 for 10 min. Blocking was performed using PBS with 5% normal goat serum for 1 h at room temperature, followed by incubation with rabbit anti-insulin antibody (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-glucagon antibody (Abcam, Cambridge, UK) diluted 1:100 at 4°C overnight. Three consecutive washes with PBS for 5 min each were followed by sequential incubation with Alexa Fluor 595 and 488 goat anti-rabbit and anti-mouse IgG secondary antibodies (1:200) at room temperature for 1 h. The slides were washed three times with PBS and mounted using an anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were captured under a fluorescence microscope (Carl Zeiss AG; Oberkochen, Germany). Islet area was determined from at least five different islets per pancreas section stained with H&E using Image J software (National Institute of Health, NIH Version v1.32j).