Rabbit anti insulin antibody

Pancreatic tissues were fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Serial 4-μm sections were mounted on slides. Localization of insulin and cleaved caspase-3 in pancreatic islets was performed by a double-labeled immunofluorescence method. The slides were deparaffinized with sequential changes in xylene and rehydrated with descending concentrations of ethanol. This was followed by antigen retrieval, conducted by boiling the slides in citrate buffer followed by gradual cooling. After being washed in PBS, the sections were treated with a blocking agent (0.5% bovine serum albumin, Sigma, USA) for 45 min at room temperature. Sections were incubated overnight at 4 °C with a cocktail of two antibodies: rabbit anti-insulin antibody (1:50, Abcam, USA) and rat anti-cleaved caspase-3 antibody (1:20, Abcam, USA). Then, sections were incubated with FITC and Cy3-conjugated secondary antibody after washing in PBS. Sections were viewed with a fluorescence microscope (Thermo, USA). The intensity of cleaved caspase-3-positive signals in the insulin-positive area was measured using Image J software.

Subcutaneous (SubQ) fat and liver tissues were fixed in 10% neutral-buffered formalin for 24 h and sectioned at 4-μm. H&E staining was performed according to the standard protocols. For Oil Red O staining, liver tissue was fixed in 1% formalin for 2 h and embedded in OCT compound. The tissue was sectioned at 10-µm and stained with Oil red O according to standard methods. For immunohistochemical analysis, pancreatic tissue was fixed in 1% formalin for 2 h and embedded in OCT compound. Sections were treated with a blocking agent (0.5% bovine serum albumin, Sigma-Aldrich, USA) for 45 min at room temperature. Sections were incubated overnight at 4 °C with rabbit anti-insulin antibody (1:50, Abcam, USA) and mouse anti-NMDAR1 antibody (1:50, Abcam, USA). Subsequently, sections were incubated with FITC-conjugated secondary antibody and were observed under a fluorescence microscope (Thermo, USA). The score of liver steatosis was according to the grade of the lesion, slight (0.5), mild (1), moderate (2), severe (3), profound severe (4), and normal (0).