Ab221549

Expanded islet clusters were harvested using cell recovery solution (Corning, 354253) and fixed in 4% paraformaldehyde for 30 min at room temperature, followed with blocking and permeabilizing in PBS with 0.5% Triton X-100 (Solarbio, T8200) and 5% donkey serum (Solarbio, SL050) for 30 min at room temperature. Then, samples were incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. DAPI (Beyotime, C1002) was used to stain the nucleus and find islets. The following antibodies were used for immunofluorescence: anti-insulin (1:200, sc-9168; Santa), anti-somatostatin (1:600, ab30788; Abcam), anti-glucagon (1:200, G2654; Sigma). anti-PDX1 (1:200, ab47267; abcam), anti-SOX9 (1:200, ab185966; abcam), anti-NKX6.1 (1:200, ab221549; abcam), anti-MAFA (1:200, ab26405; abcam), anti-KI67 (1:200, D3B5; Cell Signaling Technology). Imaging of the expanded islet clusters was performed on Zeiss LSM 780 and processed using ImageJ or Adobe illustrator software.

Cells from each stage of the groups described above were fixed in 4% paraformaldehyde for 20 min. After washing with PBS, the cells were permeabilized with 0.1% Triton-100 for 20 min, and then blocked with goat serum for 30 min. They were then incubated with the corresponding primary antibodies, which included Pdx1 antibody (rabbit, 1:200, #5679S, CST). C-peptide antibody (rabbit, 1:100, #4593S, CST), Foxa2 antibody (Mouse, 1:200, #ab108422,Abcam), Sox17 antibody (Mouse,1:100, #bs12205R, Bioss), MAFA antibody (rabbit, 1:200,#bs0924R, Bioss), GLUT2 antibody (rabbit, 1:200, #bs1010379R, Bioss), Nkx6.1 antibody (rabbit, 1:200, #ab221549, Abcam), and insulin antibody (rabbit, 1:200, #ab181547, Abcam) overnight at 4 ℃. Next, the cells were washed three times with PBST and further incubated for 1 h in a wet box in the dark with the following secondary antibodies: that included goat anti-rabbit immunoglobulin G heavy and light chain antibodies (IgGH&L, 1:250, Proteintech) and anti-mouse IgG (H + L), F(ab’)₂ fragment (Alexa Fluor® 555 Conjugate). The nuclei were stained with Hoechst 33342 for 7 min after washing with PBST and were then observed under a fluorescence microscope (NOVA View^R).