Planapo360 immersion oil objective

HUVEC cells cultured on coverslips were stimulated with human MMP-9 for 5 minutes, as described above. The cells were then washed with HBSS without Ca²⁺ and Mg²⁺, fixed with 100% methanol, blocked with 5% BSA in PBS followed by incubation with anti-PAR-1 receptor antibody (ATAP-2, Zymed Laboratories Inc.; Bedminster, NJ). Anti-PAR-1 (ATAP-2) antibody recognizes intact and cleaved forms of PAR-1 receptor. Finally, cells were incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen; Waltham, MA). Cleavage of PAR-1 receptor was visualized using Perkin Elmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope and PlanApo360 immersion oil objective (numerical aperture 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.

HUVEC cells cultured on coverslips were incubated for 5 minutes with human MMP-9 (Calbiochem; San Diego, CA) at the concentration range of 0.1–0.01 nM in medium without serum containing sodium orthovanadate (1 mM) (Sigma Aldrich; St. Louis, MO). In some experiments HUVEC cells were treated with monoclonal antibodies against PAR1 receptor (WEDE15 and ATAP2; Immunotech Laboratories Inc.; Monrovia, CA and Santa Cruz Biotechnology; Dallas, TX, respectively) for 30 minutes at 4°C prior to incubation with human MMP-9. After 5 minutes of stimulation with human MMP-9, the cells were washed with HBSS without Ca²⁺ and Mg²⁺, fixed with 100% methanol, permeabilized with 0.5% (vol/vol) Triton X-100 in PBS, blocked in 5% BSA in PBS, and incubated overnight at 4°C with anti-pERK (Thr202/Tyr204) antibody (Cell Signaling Technology; Danvers, MA). To visualize phosphorylation of ERK, HUVEC cells were then incubated with Alexa Fluor 488 conjugated chicken anti-rabbit antibody (Invitrogen; Waltham, MA). The cells were observed using Perkin Elmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope and PlanApo360 immersion oil objective (numerical aperture [NA] 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.