Alexa568 conjugated isolectin

GFP-labeled Tie2-Akt-ECs were intrasplenically injected into C57BL/6 mice (n=4) immediately following 70% partial hepatectomy, as previously described [36 (link)]. Three weeks after injection of the GFP-labeled Tie2-Akt-ECs, intravital labeling of the vasculature was achieved with Alexa568-conjugated Isolectin (Invitrogen) or anti-VE-Cadherin (Biolegend). The mice were then sacrificed, and their livers were harvested, processed, and analyzed as the Matrigel plugs above.

Control, ER71- or Akt-transduced Tie2-ECs were labeled with a PGK-GFP lentivirus. These cells were mixed with Matrigel (BD), then injected subcutaneously to the flanks of NOD-SCID-IL2Rγ^−/− (NSG) mice (Jackson Laboratories, ME) (n=5). After two weeks, intravital labeling of the vasculature was achieved with Alexa568-conjugated Isolectin (Invitrogen) (2mg/kg). After 10 minutes, the Matrigel plugs were harvested, fixed in 4% paraformaldehyde overnight, and saturated with 30% sucrose for 48 hours. 30µM sections were stained with PECAM (BD), then counterstained with DAPI. Confocal images were obtained on a Zeiss 710 META confocal microscope (Carl Zeiss, Germany).