Anti c peptide

Immunofluorescence staining was performed as previously reported^{10 (link)}. The following primary antibodies were used: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-C-peptide (dilution 1:100, #4593S; Cell Signaling Technology), anti-PDX-1 (dilution 1:200, #5679; Cell Signaling Technology), anti-NKX6.1 (dilution 1:400, #54551; Cell Signaling Technology), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech, Chicago, IL, USA), anti-GAD65 (dilution 1:50, ab239372, Abcam plc.) and anti-PD-L1 (dilution 1:25, 17952–1-AP, Proteintech). Anti-rabbit Alexa 488 (A-11008; Thermo Fisher Scientific Inc.) for anti-C-peptide, PDX-1, ICA, ZnT8, GAD65 and PD-L1 antibodies, and anti-mouse Alexa 594 (A-11005; Thermo Fisher Scientific Inc.) for anti-insulin antibody were used as the secondary antibodies. 4′, 6-diamidino-2-phenylindole (DAPI) (P-36931; Thermo Fisher Scientific Inc.) was applied for nucleus staining.

For immunohistochemical examination, mice at 15~16 weeks of age were perfused with 4% paraformaldehyde and subjected to sectioning using Cyrostat (Leica). Pancreatic sections were stained using anti-Insulin (HyTest), anti-C peptide (Cell Signaling), anti-ERK, anti-Phospho-ERK (Cell Signaling), anti-CREB anti-Phospho-CREB (Cell Signaling), and anti-glucagon (Sigma-Aldrich) antibodies. All antibodies were used at 1:500 dilutions. DAPI solution (Dojindo) was used to stain nuclei. Images were obtained using a FV1000 confocal microscope (Olympus). For measurement of the islet area, randomized pancreatic sections were obtained from 3 mice, and stained with anti-insulin and anti-glucagon antibodies. The insulin-positive and glucagon-positive areas were measured as islet areas using Image J software (NIH), as described previously37 (link).

After 72 h of transfection, cells were gently washed with 0.01 M PBS (phosphate-buffered saline, pH 7.4), fixed with 4% paraformaldehyde (in PBS) for 20 min at room temperature and then permeabilized with 0.3% Triton X-100 (in PBS) for 13 to 17 min. After blocking with 10% horse serum for 50 min at 37℃, cells were incubated with a primary antibody against PDX1 (cat# ab47383, Abcam, Cambridge UK) or anti-C-peptide (cat# 4593s, Cell Signaling Technology, MA, USA), which had been diluted in 10% horse serum (in PBS), overnight at 4℃. Next, the cells were incubated with secondary antibody diluted in 1% BSA for an hour at 37℃. Cell nuclei were stained with Hoechst 33342 (cat# C1022, Beyotime, Shanghai, China) for 5 min at room temperature. Images were captured using DeltaVision OMX (General Electric Company, Fairfield, Connecticut, USA).