Alexa 488 conjugated goat anti mouse igg monoclonal antibody

Cell monolayers were seeded onto microscope cover glass. After 30 min of treatment, the cells were fixed in 4% paraformaldehyde for 5 min followed by three washes with PBS. The cells were then blocked with Bløk-FL noise canceling reagent (Millipore) for 1 h at room temperature. To detect VE-cadherin localization, a mouse anti-VE-cadherin monoclonal antibody (Beckman Coulter, Brea, CA) (1:200 dilution in PBS) was incubated with the cells overnight at 4°C. After three washes with TBST, the cells were treated with an Alexa 488-conjugated goat anti-mouse IgG monoclonal antibody (Invitrogen, Carlsbad, CA) (1:500 dilution) and Alexa Fluor 647 Phalloidin dye (Invitrogen) (1:1,000 dilution) for 1 h. Subsequently, three washes were performed with Tris-buffered saline-tween 20. Images were obtained using a confocal microscope (Olympus FluoView FV1000, Melville, NY).

Cell monolayers were seeded onto microscope cover glass. After treatment, the cells were fixed in 4% paraformaldehyde for 5 min, followed by three washes with PBS. The cells were then blocked with SuperBlock blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. To detect VE-cadherin and LC3 localization, specific antibodies against VE-cadherin (Beckman Coulter, Brea CA), and LC3 (Genetex) (1:200 dilutions in PBS) were incubated with cells overnight at 4°C. After three washes with tris-buffered saline and Tween 20, the cells were treated with an Alexa 488-conjugated goat anti-mouse IgG monoclonal antibody (Invitrogen, Carlsbad, CA) (1:500 dilution) and Alexa 594-conjugated goat anti-rabbit pAb (Invitrogen) (1:1,000 dilution) for 1 h, followed by three washes with tris-buffered saline-Tween 20. Images were obtained using a confocal microscope (Olympus FluoView FV1000, Melville, NY).
For quantifying barrier/marginal VE-cadherin, 3 μm across the cell border was defined as barrier/marginal area. And the remaining area within a cell was defined as cytosolic/perinuclear area. 50 cells in each condition were quantified by using Image J software.