Alexa488 conjugated antibody

For immunocytochemistry, HeLaS3 cells transfected with the 1 μg of TDP-43 (WT, G348C or A382T) /pFLAG-CMV2 fixed with 4% paraformaldehyde (PFA) for 5 min at room temperature (RT) and washed with PBS after 24 h of transfection, and permeabilized with 0.01% Triton X-100 for 15 min at RT. After washing with PBS and incubation in 5% BSA for one hour at RT, cells were incubated with the primary antibodies, anti-DDDDK-tag (MBL M185-3 1:1000), for overnight at 4 °C. The cells were washed with PBS, then incubated with the secondary antibody Alexa 488-conjugated antibody (Molecular Probes A11001, 1:200) and Hoechst 33342 (Molecular Probes H3570, 1:1000), for one hour at RT. After washing with PBS, fluorescence was observed using a confocal laser scanning microscope (Zeiss) with a Plan Apochromat 20×/0.8 objective. For promoter reporter overexpression and nuclear staining, HeLaS3 cells transfected with 1 μg of the Venus reporter vector (driven by CMV promoter or P1) were fixed with 4% PFA for 5 min RT and washed with PBS after 48 h of transfection. Incubation with Hoechst 33342 was performed for one hour. After washing with PBS, fluorescence was visualized using the confocal microscope with a Plan Apochromat 63×/1.4 oil immersion objective. Comparison images were acquired with the same laser intensity, exposure time, and filter. Details are described in each figure legend.

Human umbilical vein endothelial cells on PET membrane transwell inserts from the above experiment were fixed with 4% formaldehyde, and incubated with mouse antihuman vascular endothelial (VE)–cadherin antibody (1:100; Abcam), followed by incubation with secondary goat anti-mouse Alexa 488–conjugated antibody and Alexa 633–conjugated phalloidin (1:100) and DAPI for nucleus (1:100; Molecular Probes). After incubation with each antibody, the membranes were washed three times for 10 min with BD Permwash. Finally, membranes were dipped once in water and mounted with Fluorosave (Calbiochem) on microscopic glass slides. The membranes were then visualized under a Zeiss-510 confocal microscope at a magnification of 100×.

Coverslips were seeded with 3 × 10⁴ HUVEC per slide and cultured for 48 h. Cells were then fixed and immuno-stained with an anti-Vascular Endothelial Cadherin antibody (R&D Systems) followed by appropriate Alexa-488-conjugated antibody (Molecular probes). Cells were also counterstained with TRITC-conjugated phalloidin and Hoechst (Sigma-Aldrich, Dorset, UK) and imaged as before. Cells that possessed filopodia or lamellipodia were counted as “activated”, while cells without these structures and those that demonstrated peripheral distribution of the actinic cytoskeleton were counted as “quiescent” using the Cell Sense software counting tool (manual, computer-aided counting). The ratio between “activated” and “quiescent” HUVEC for each treatment was calculated and represented in a Log₁₀ scale.