Axiocam mrm monochrome color camera

Primary hRECs and HUVECs were fixed in 4% PFA for 10 min and blocked in 3% BSA with 0.1% T-PBS (permeabilized) or with PBS (non-permeabilized) for 1 hr. Samples were incubated with primary antibodies overnight at 4°C, then with secondary antibodies at room temperature for 2 hr. Primary antibodies were rabbit anti-ezrin (1:200), rabbit anti–pERK (1:100), rabbit anti–neuropilin 1 (1:200), rabbit anti–CD31 (1:200), mouse anti-myc epitope tag (1:500), Alexa Fluor™ 594 Phalloidin (1:200), and goat anti–VEGFR2 (1:200), mouse anti-myc epitope tag (1:500; Cell Signaling Technology). Secondary antibodies were goat/donkey anti-mouse or donkey anti-rabbit conjugated to Alexa Fluor 488 or 594 (Invitrogen) used at a 1:500 dilution. Specimens were mounted in 50% glycerol, 50% PBS, and 0.04% sodium azide and viewed using an Axioskop 2 Mot Plus microscope equipped with an Axiocam MRm monochrome color camera (Carl Zeiss Inc.). Experiments were performed at least three times using independent cell samples.

Primary hRECs at 70% confluence were treated with eVEGF-38/AAV2, eVEGF-53/AAV2, VEGF189/AAV2 or GFP-Ctr/AAV2 individually at 50 MOI each. Three days later, the cells were fixed and staining for the expression of transgenes (anti-myc antibody, Cell Signaling Technology) and GFP (auto fluorescent or anti-GFP antibody, company, city, state), and photographed under an immunofluorescence microscope (Axioskop 2 Mot Plus microscope equipped with an Axiocam MRm monochrome color camera, Carl Zeiss Inc.) for determining the transduction efficiency. Cells were used for drug treatment, immunostaining and western blotting.