Rabbit anti dll4

Samples were fixed in 4% PFA for 20 min at RT. Samples were then quenched with 100 mM glycine, washed once with PBS, and were then blocked for 1 h with 1% BSA (w/v) at RT. Mouse anti-CD31 [1:20 in 1% BSA (w/v); Dako, United Kingdom] and rabbit anti-DLL4 [1:200 in 1% BSA (w/v); Abcam] primary antibodies were added before incubating at RT overnight. The samples were subsequently washed for 2 h in 1% BSA/0.1% Tween 20 (w/v/v/v), followed by incubation with Alexa Fluor 546 goat anti-mouse IgG secondary antibody (Life Technologies) for 2 h at RT. Nuclear counterstaining was performed using DAPI by incubating at RT for 20 min. Finally, the samples were washed with 1% BSA/1% Tween20 for at least 8 h with hourly changes of the BSA/Tween20 solution. Samples were imaged using a Zeiss LSM 510 confocal microscope (Zeiss).