Mouse anti neurod1

Five days after gene transduction, the expression of insulin and glucagon was checked in DIPCs. Guinea pig anti-insulin and mouse anti-glucagon (1:1000; Abcam, Cambridge, UK) were used as primary. To determine the expression of transduced pancreatic endocrine transcription factors, we used rabbit anti-PDX1, mouse anti-NEUROD1, and rabbit anti-MAFA (1:200; Abcam, Cambridge, MA, USA). Samples were incubated overnight with primary antibodies at 4 °C. For secondary fluorescence labeling, cells were incubated with anti-guinea pig IgG Alexa Fluor 647 (1:200; Abcam, MA, USA), anti-rabbit IgG Alexa Fluor 488 (1:200; Thermo Fisher Scientific, Waltham, MA, USA), and anti-mouse IgG Alexa Fluor 488 (1:200; Thermo Fisher Scientific). Finally, cells were mounted using prolong gold antifade mountant containing DAPI (4′,6-Diamidino-2-phenylindole) (Life Technologies, Frederick, MD, USA). The slides were visualized under an EVOS FL auto cell-imaging system (Thermo Fisher Scientific). For DIPC spheroid staining, spheroids were harvested and fixed with 4% paraformaldehyde, processed in 30% sucrose for 3 days, and then embedded in optimum cutting temperature compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan). Cryostat sections (6 μm) were sliced, and the subsequent process was the same as that for DIPCs.

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After antibody blocking, primary antibodies were incubated overnight at 4 °C. We used rabbit anti-insulin and mouse anti-glucagon (1:1000; Abcam, Cambridge, UK), rabbit anti-PDX1, goat anti-PDX1, mouse anti-NEUROD1, rabbit anti-MAFA (1:200; Abcam), and mouse anti-albumin (1:100, Santa Cruz Biotechnology). For secondary fluorescence labeling, the cells were incubated with anti-rabbit IgG Alexa Fluor 488, anti-rabbit IgG Alex Fluor 555, anti-goat IgG Alex Fluor 488, anti-mouse Alexa 555, and anti-mouse Alexa 488 (1:200; Thermo Fisher Scientific). ProLong Gold antifade reagent containing DAPI (Thermo Fisher Scientific) was used to stain the nuclei and for mounting. The slides were visualized using the EVOS® FL auto cell imaging system (Thermo Fisher Scientific). Under the microscope, the cells with red fluorescence were insulin or glucagon positive and blue fluorescence highlighted the nucleus and counted in 10 randomly selected fields per IPC sheet or IPC cells with total of 3 donors in each group. The results were presented as insulin- or glucagon-positive cells per 100 cells.