Rat anti mouse pecam 1

For whole-mount immunostaining, tissue was fixed in 4% PFA for 2h at RT, permeabilised in 0.3% Triton-X100 in PBS (PBSTx) and blocked in PBSTx plus 3% milk. Primary antibodies were incubated at 4C overnight in blocking buffer. After washing in PBSTx, the samples were incubated with fluorocrome-conjugated secondary antibodies in blocking PBSTx plus 1% milk, washed and mounted in Mowiol. The following antibodies were used: rat anti-mouse LYVE-1 (R&D systems), rat anti-mouse PECAM1 (BD), rabbit anti-human Prox1 (generated against human Prox1 C-terminus (567-737aa), Prox1-GST construct provided by Dr. T. Petrova, University of Lausanne), rabbit anti-GFP (Invitrogen), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (Santa Cruz Biotechnology), hamster anti-mouse Podoplanin (Developmental Studies Hybridoma Bank), goat anti-mouse Neuropilin-2 (all from R&D Systems). Secondary antibodies conjugated to DyLight 405, AF488, Cy3 or AF647 were obtained from Jackson ImmunoResearch. For primary LECs immunostaining, cells were fixed with 4 % PFA/PBS for 20 min at RT and permeabilized using 0,5% Triton-X-100/PBS for 5 min at RT followed by blocking with 3% BSA/0.3%Triton for 1h and incubation with primary antibodies (GFP-antibody; VE-cadherin C19, SantaCruz). Primary antibodies were detected with AlexaFluor 488 and AlexaFluor 647 -coupled secondary antibodies (Jackson Immunoresearch).