HUVECs were transferred from a 100 mm dish into a 6-well plate after transfection or infection. Cells were fixed with 4% PFA in RT for 10 min. After 2 rounds of brief washing with PBS, permeabilization/blocking solution (1% BSA with 0.2% triton x-100 in PBS) was added to the fixed cells for 30 min at RT. Rabbit anti-Paxillin (Cell Signaling, 1:100) and mouse anti-GFP (Chemicon, 1:100) in PBS were administered for 1 h at RT. Following three washes with PBS (5 min each), suitable species-specific Alexa Fluor-coupled secondary antibodies (1:500) were administered togehter with 1:1000 DAPI and samples were incubated for 25 min at RT. After three washes with PBS (5 min each), stained cells were mounted on microscope glass slides with Fluoromount-G (SouthernBiotech, 0100-01).
For labeling of proliferating cells, 100 μg of EdU (Life Technologies) per pup was injected intraperitoneally 4 h before sacrifice. EdU-positive cells were detected with the Click-iT EdU Alexa Fluor-647 Imaging Kit (Life Technologies, C10340).
For labeling of proliferating cells, 100 μg of EdU (Life Technologies) per pup was injected intraperitoneally 4 h before sacrifice. EdU-positive cells were detected with the Click-iT EdU Alexa Fluor-647 Imaging Kit (Life Technologies, C10340).