Anti insulin pab

Handpicked pancreatic islets were seeded on SuperFrost Gold Plus microscope slide by cytospin. Then, islets were fixed, permeabilized, and after stained with anti-insulin pAb (Abcam), anti-glucagon mAb (Abcam) and anti-CRAMP pAb or anti-LL-37 pAb (Innovagen), overnight at 4 C. After washing, second-step reagents were applied: anti-guinea pig-AlexaFluor488 (insulin), anti-mouse-Alexa647 (glucagon), and anti-rabbit-AlexaFluor555 pAbs (CRAMP and LL-37) (Invitrogen). Nucleuses were stained with DAPI. Image acquisition was performed on Necker Institute Imaging Facility using a Leica SP8 confocal microscope.
In Vitro Treg Cell Induction All cells were magnetically isolated using MACS cell separation system (Miltenyi). CD62L + CD4 + CD25 À BDC2.5 T cells (4 3 10 4 cells per well) from splenocytes of BDC2.5 TCR transgenic NOD mice were incubated with 2 3 10 3 myeloid cells (CD11b + ) obtained from pancreatic islets of NOD mice treated with CRAMP or vehicle. Culture were performed for 4 days in complete IMDM with 5 ng ml À1 recombinant human IL-2 (R&D), 1 ng ml À1 recombinant mouse TGF-b (R&D) and 20 ng ml À1 of peptide 1040-51, a mimotope of BDC2.5 T cells. In some conditions retinoic acid inhibitor LE135 (1 mM, Santa Cruz) was added to the culture.