Fluoromount g

Pancreatic tissues from all mice at day 30 post diabetes induction were harvested, formalin-fixed, and sliced in 5 μm sections while BMSC for immunocytochemistry were fixed in 10% formalin overnight. For histology, tissue sections were deparaffinized with grading xylene and ethanol grades and rehydrated in water. Both tissues or cells were permeabilized and blocked with 4% donkey serum (Sigma Aldrich, USA) for 1 h at RT, followed by primary antibodies (see Table 1 for details) incubation overnight at 4 °C. The next day, cells were washed and labeled with secondary antibodies (see Table 2 for details) for 30 min at RT. Nuclei were marked with DAPI and mounted with Fluoromount-G (VECTASHIELD, USA). Images were captured on LSM710 confocal microscope and analyzed using Zen10 software (Carl Zeiss, USA).
Table 2

List of secondary antibodies

Sr. no.	Antibody	Company and catalog no.	Isotype IgG	Mono/polyclonal Ab	Application	Dilution
1	Anti-Mouse-IgG-HRP	Jackson ImmunoResearch #115-035-003	Goat	Poly	Western	1:5000
2	Anti-Rabbit-IgG-HRP	Jackson Immuno Research #111-035-003	Goat	Poly	Western	1:5000
3	Anti-Mouse-IgG-FITC	Sigma#F8771	Goat	Poly	IF	1:200
4	Anti-Rabbit-IgG-FITC	Sigma#F9887	Goat	Poly	IF	1:200
5	Anti-Mouse-IgG-CF555	Sigma#SAB4600299	Goat	Poly	IF	1:100
6	Anti-Rabbit-IgG-CF555	Sigma#SAB4600068	Goat	Poly	IF	1:100

Cells were fixed in paraformaldehyde 4% for 20 min. After paraformaldehyde quenching (NH₄Cl 50 mM), permeabilization (Triton X-100 0.2% in PBS) and blocking (bovine serum albumin 3% in PBS) steps, cells were incubated 1 h in primary antibodies (rabbit anti-EEA1 primary antibody C45B10, Cell Signaling, 1/500; mouse anti-MAP 2 antibody, MAB3418, Millipore, 1/500; mouse anti-Phosphatidylinositol-3-phosphate (PI (3) P) antibody, Z-P003, Echelon Biosciences, 1/200). Cells were rinsed and incubated in anti-rabbit AlexaFluor-488 and anti-mouse AlexaFluor-568 secondary antibodies for 1 h (1/1000, Invitrogen), counterstained with DAPI (1 μg/mL, Vector Laboratories), rinsed and mounted in Fluoromount-G for confocal microscopy or Vectashield (Vector Laboratories) for SIM. For confocal microscopy, z-stacks images (1024 × 1024 pixels, representing voxels of 0.0451 × 0.0451 × 0.198 μm) were taken using a Leica TCS SP8 AOBS confocal microscope with a 63x/NA = 1.40 oil immersion objective and × 4 zoom at ICMQuant facility. For fibroblasts, we analyzed between 26 and 32 cells in each individual, in a total of 3 euploid individuals and 3 individuals with DS.