Alexa fluor 488 conjugated anti rabbit or anti mouse

The samples on the slides were fixed with 4% formaldehyde for 20 min at room temperature. We gave the slides three times wash with PBS, followed by permeabilization with 0.3% Triton X-100 for 30 min, rewashing three times, and blocking in 10% goat serum in PBS for 2 h at room temperature. The following antibodies were used for immunostaining: PDGFR-β (1:1000; ab32570; Abcam) and CD31 (1:1000; ab24590; Abcam). The slides were washed three times with PBS, followed by incubation with Alexa Fluor 488–conjugated anti-rabbit or anti-mouse (Invitrogen) for 1 h at room temperature. After a final washing with PBS three times, the slides or coverslips were mounted using Prolong Gold Antifade Reagent (Invitrogen). Fluorescent images were taken at room temperature on a Zeiss Observer, under the condition of a Z1 inverted microscope with a 40 × /1.3 or 63 × /1.4 oil-immersion objective. The images were analyzed with ImageJ software.

Islet-like aggregates were detached and single cells disaggregated by trypsin treatment. Next, cells were fixed by 2% paraformaldehyde for 10 min and recovered by centrifuge (1000 rpm for 5 min). After washing twice with PBS, cells were permeabilized with PBS containing 1% of Tween-20 for 10 min at RT, washed again twice with PBS, and incubated with blocking solution (2% BSA, 10% FBS in PBS) for 10 min. After, cells were incubated for 1 h at RT with the following antibodies: Guinea pig anti-human insulin, rabbit anti-human glucagon (Linco Research, St. Charles, MO, USA); rabbit anti-human PDX-1, rabbit anti-human cytokeratin19 (CK-19; Millipore, Billerica Headquarters, Billerica, MA, USA); rabbit anti-human Nkx2.2, rabbit anti-human Nkx6.1 (S. Cruz Biotech. Inc., Heidelberg, Germany); and mouse anti-human MUC-1, mouse anti-human somatostatin (Ab-Serotec, Oxford, UK). Then cells were washed three times with PBS and incubated with the appropriate fluorescent secondary antibodies: PE-conjugated anti-Guinea pig (Jackson Lab.), Alexa Fluor 488-conjugated anti-rabbit or anti-mouse (Invitrogen). Isotype identical antibodies served as control. Then, cells were promptly analyzed with a BD FACSCalibur equipped with the CellQuest software.