Ab7760

Cells were seeded at a density of 1X10⁵ /well on an 8-well chamber slide and fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at RT. Then the cells were permeabelized using 0.2% Triton X-100 for 5 min at room temperature and incubated overnight with primary antibody at 4°c. The primary antibodies were mouse anti-human insulin (1:100) (# ab7760, Abcam, Cambridge, MA, UK), mouse anti-human Glucagon (1:200) (#ab10988), mouse anti-human PDX1 (1:200) (#ab84987) and mouse anti-human Neurogenin3 (1:100) (#ab87108). After washing with PBST 3 times, the slides were incubated with fluorescence-labeled secondary antibody, including FITC-coupled goat anti-mouse IgG (1:50) (#AF8032, Razi Biotech, Iran) and Texas Red-labeled goat anti-mouse IgG (1:100) (ab5884) for 45 min at RT away from dark. Nuclear DNA was stained with 0.1μg/ml of blue-fluorescent 4', 6-Diamidino-2-phenylindole (DAPI) (Sigma) at 30°C for 5 min after washing with PBST 3 times. The slides were mounted with glycerol-PBS and visualized under a fluorescence microscope (Nikon.US). All experiments were repeated at least two times. In addition, pancreatic endocrine markers checked by western blot analysis.

For immunocytochemical analysis, differentiated cells were scraped at day 21 and cells were washed twice in cold 2% FBS-PBS, then diluted in 100 ml of cold 1% bovine serum albumin in phosphate-buffered saline (BSA-PBS). 105 cells were fixed on slides by cytocentrifugation (Shandon Cytospin, USA). Cells were coated on the slides and fixed with 4% paraformaldehyde (PFA, Merck, Germany) for 20 minutes at room temprature. Then, cells were permeabilized with 0.2% Triton (Merck, Germany) 100-X for 5 minutes. Slides were washed twice and incubated in 5% goat serum for 45 minutes. The primary antibody were mouse anti-human insulin (1:100# ab7760, Abcam, Cambridge, MA, USA), mouse anti-human Neurogenin3 (1:100# ab87108 Abcam, Cambridge, MA, USA) and mouse anti-human PDX1 (1:200#ab84987 Abcam, Cambridge, MA, USA). Cells were washed 3 times with PBS with Tween 20 (PBST) and incubated for 45 minutes at room temperature with fluorescence-labeled secondary antibody, including fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (1:50, #AF8032, Razi Biotech, Iran) and Texas red-labeled goat anti-mouse IgG (1:100, #ab5884). Nuclear DNA was stained with 0.1 μg/ml of blue-fluorescent 4΄, 6-Diamidino-2-phenylindole (DAPI, Sigma, USA) at 30˚C for 5 minutes. The slides were visualized under a fluorescence microscope (Nikon, USA).

Isolated mouse pancreata were fixed using Mildform (Wako) overnight at 4°C and embedded in paraffin (Sakura Finetek Japan). Sections of 4 μm were treated with 0.2% Triton-X100/PBS for 5 min and incubated with boiled 0.01 M citrate buffer, pH 6.0, for 20 min. Prior to application of primary antibody, sections were treated with Blocking Solution A from the Histofine MOUSESTAIN kit (Nichirei). Primary antibodies, including anti-OPG rabbit polyclonal antibody at 5.0 μg/ml (ab73400, abcam), anti-insulin mouse monoclonal antibody IN-05 at 1.0 μg/ml (ab7760, abcam), and anti-glucagon mouse monoclonal antibody K79bB10 at 4.4 μg/ml (ab10988, abcam) were applied overnight at 4°C. After washing with PBS, cells were stained with Alexa 488-conjugated goat anti-mouse IgG or Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes) for 1 hr at room temperature. After washing, sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and observed under an LSM710 confocal fluorescence microscope (Carl Zeiss) to determine signal localization or under a DMi6000B microscope (Leica) to measure signal intensity. The average signal intensity was measured by freehand region of interest (ROI) using Tissue Studio (Definiens) software to quantify insulin expression levels in pancreatic β-cells.