Anti glucagon antibody

Paraffinized sections were deparaffinized and rehydrated, and antigen was retrieved with 10 mM Tris/EDTA (pH 9.0). The sections were permeabilized and blocked in PBS containing 1% BSA and 5% goat serum. Primary antibodies were incubated overnight at 4°C, followed by secondary antibodies incubation at 37°C for 1 hour. For immunofluorescence staining, anti-insulin antibody (Abcam, Cambridge, UK) and anti-glucagon antibody (Abcam) were used to detect β and α cells, respectively. Pancreatic sections were stained with anti-insulin antibody (HUABIO, Hangzhou, China) and anti-PCNA antibody (HUABIO), followed by tyramine amplification with fluorophores 488 and 594 and DAPI counterstaining to determine the proliferation of β cells. Insulin-positive (insulin+) cells showing nuclear colocalized staining for DAPI+ and PCNA+ were considered as proliferating β cells. Sections were visualized using a confocal microscope (LSM 880; Carl Zeiss, Oberkochen, Germany).
For immunohistochemistry staining, pancreatic sections were incubated with anti-insulin antibody (Abcam), followed by binding with horseradish peroxidase (HRP)-conjugated secondary antibodies, and detected with a DAB Kit and counterstained with hematoxylin. Quantification was performed using ImageJ.

Insulin and glucagon contents in pancreatic islets were determined through an immunohistofluorescence analysis. Briefly, fresh pancreatic tissues were fixed in 10% paraformaldehyde and embedded prior to sectioning. All sections were incubated with secondary antibody (Jackson ImmunoResearch, USA) in the dark for 1 hr at room temperature and dyed with DAPI after staining with primary antibody (anti-insulin antibody (CST, USA) and anti-glucagon antibody (Abcam, USA)) at 4°C overnight.
The insulin content was measured, and Stat3 translocation in INS-1 cells was detected through an immunocytofluorescence analysis. INS-1 cells were seeded on circular slides in a 6-well plate. After the corresponding treatment, the cells were fixed in 4% paraformaldehyde for 15 min and blocked with 5% BSA for 30 min, followed by incubation with the corresponding primary antibody (anti-insulin antibody (CST, USA) and anti-Stat3 antibody (Abcam, USA)) at 4°C overnight, and the cells were then incubated with FITC-tagged secondary antibody (Jackson ImmunoResearch, USA) in the dark for 1 hr at room temperature and dyed with DAPI to capture the distribution of fluorescence under a fluorescence microscope (Olympus, Japan).

Anti-F4/80 antibody (AbD Serotec), anti-alpha 1 sodium potassium (NaK) ATPase antibody (Abcam), anti-HIF-1α antibody (Cayman Chemical), anti-HIF-2α antibody (Novus Biologicals), anti-HIF-1β antibody (Novus Biologicals), anti-α-tubulin antibody (Sigma), anti-insulin antibody (Cell Signaling), and anti-glucagon antibody (Abcam) were used.