Mouse anti ve cadherin monoclonal antibody

A monolayer of cells was seeded onto a microscope cover glass slide. After 30 min of treatment, the cells were fixed in 4% paraformaldehyde for 15 min followed by three washes with PBS. The cells were subsequently blocked with Superblock T20 (PBS) Blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. To detect VE-cadherin, LC3 puncta localization, or the expression of the MIF receptors, a mouse anti-VE-cadherin monoclonal antibody (Beckman Coulter), a rabbit anti-LC3 polyclonal antibody (GeneTex), a goat anti-CD74 polyclonal antibody (SantaCruz), a rabbit anti-CXCR4 polyclonal antibody (GeneTex), or a rabbit anti-CXCR7 (GeneTex) polyclonal antibody (1:200 diluted in PBS) were incubated with the cells overnight at 4°C. After three washes with Tris-buffered saline-Tween 20 (TBST), the cells were incubated with an Alexa 488-conjugated goat anti-mouse IgG secondary antibody, an Alexa 594-conjugated goat anti-rabbit IgG secondary antibody or an Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, Calif) (1:500 diluted) and Hoechst 33342 (Invitrogen) (1:3,000 diluted) for 1 h, and the slides were washed 3 times with PBS. The images were acquired using a confocal microscope (Olympus FluoView FV1000, Melville, NY).