Mouse anti human cd31

At the endpoint of in vitro culture, mouse and human islets were fixed in 4% paraformaldehyde for 15–30 minutes, washed in PBS and directly stained (mouse islets) or incubated in 20% sucrose in PBS (human islets) overnight at 4°C. Thereafter they were permeabilized in 0.3% Triton-X/PBS for 15–20 minutes and treated with either 6% fetal calf serum (mouse islets) for 1hour or 10% goat serum (human islets) for 30 minutes at RT. Immunohistochemical and immunocytochemical stainings were performed using guinea pig anti-mouse insulin (1:100, AbCam) or guinea pig anti human insulin (1:100, DAKO) followed by Alexa Flour 488-labeled goat anti-guinea pig (1:1000, Invitrogen), and rabbit anti mouse CD31 (1:50, AbCam) or mouse anti-human CD31 (1:100, BD Pharmingen) followed by Alexa flour 594-labeled goat anti-rabbit (1:1000) or Alexa Flour 545-labeled rabbit anti-mouse (1:1000), overnight at 4°C and 1h at RT, respectively. Counterstaining was performed with DAPI (1:1000; Sigma-Aldrich). The same protocol using the endothelial cell marker CD31 was also used for analysis of intra islets endothelial cells content in mouse islets directly after isolation and after 2 weeks of culture, as well as for analysis of cell growth emanating from the human islets after long term culture.

For immuno-staining, cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. Non-specific binding was blocked with 5% goat serum in PBS. The following antibodies were applied on each well independently: mouse monoclonal primary antibody against human microtubule associated protein-2 (MAP-2) or against human βIII-tubulin, rabbit anti-human tyrosine hydroxylase (a gift from Dr. Yves Sauve, University of Alberta), rabbit anti-glial fibrillary acidic protein (GFAP, 1:500), rabbit monoclonal against S100B (1:100; abcam), mouse anti-SSEA-4 (10 μg/ml; Millipore), mouse anti-human CD31 (1 μg/ml; BD Pharmingen), rabbit anti-VE-cadherin (4 μg/ml; Cayman, Cedarlane laboratories) and mouse anti-alpha-fetoprotein (1 μg/ml; abcam). After incubation with either Alexa 594- or 488- conjugated secondary antibodies the coverslips were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, molecular probes), and visualized under fluorescence microscopy.

For immunofluorescence studies, specimens were fixed for 20–25 min in 4% paraformaldehyde (PFA), rinsed in PBS, transferred into 10% and 30% sucrose in PBS, and embedded in tissue freeze medium (Tissue Tek, Sakura Finetek Zoeterwoude, NL). Sections of 12–14μm were incubated with the following primary antibodies: Rabbit-anti-human CCBE1 (1:500, Sigma-Aldrich, München, Germany), rabbit-anti-human ESAM-1 (1:100, Sigma-Aldrich, München, Germany), goat-anti-human smooth muscle α-actin (SMA) (Acris Antibodies, Herford, Germany), mouse-anti-human CD31 (1:50, BD Pharmingen), mouse-anti-human CD117 (c-KIT, 1:100, Santa Cruz Biotechnol., and 1:1, Dako, Hamburg, Germany), mouse-anti-human D2-40 (1:200, Dako, Hamburg, Germany), rabbit-anti-human Lyve-1 (1:500, ReliaTech, Braunschweig, Germany), rabbit-anti-human Prox1 (1:500, ReliaTech, Braunschweig, Germany), mouse-anti-human vimentin (1:200, Dako), mouse-anti-human PDGFRα (1:1, Dako), mouse-anti-human CD34 (1:200, Dako). Secondary antibodies were: goat-anti-mouse Alexa 488/594, goat-anti-rabbit Alexa 594, donkey-anti-goat Alexa 488 (MobiTech, Göttingen, Germany). Sections were counter-stained with Dapi and mounted under cover slips with Fluoromount-G (Southern Biotechnology, US). Photos were taken with AxioImagerZ1 (Zeiss, Göttingen, Germany).