Fluoview ver 2.0b viewer

Cells seeded on glass-bottom culture dishes (MatTek) were treated as described. After rinsing three times with 1× PBS, cells were fixed at room temperature with 4% formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in 1× PBS for 5 min. Fixed cells were then blocked in blocking buffer (1 mg/ml BSA in 1× PBS) at room temperature for 30 min and then incubated in blocking buffer with primary antibodies at room temperature for 2 h. After washing three times with washing buffer (0.1% Tween-20 in 1× PBS; 5 min per wash), cells were incubated in blocking buffer with secondary antibodies at room temperature for 1 h. Finally, cells were washed three times in washing buffer for 5 min each wash and mounted with Prolong Gold anti-fade reagent with DAPI (Molecular Probes). Confocal images were generated by confocal laser scanning microscopy using an FV1000 Fluoview (Olympus) and analyzed using Fluoview Ver. 2.0b Viewer (Olympus).

Cells were grown in glass bottom culture dishes (MatTek) for 16 h and were transfected or not transfected with fluorescently labeled ASOs for 4–6 h. For free uptake, cells were incubated with 1000-nM fluorescent-labeled ASOs for 16–24 h. Immunofluorescence staining was performed as described (26 (link)), with minor revisions. Briefly, cells were fixed with 4% formaldehyde for 30 min at room temperature and were permeabilized for 5 min using 0.15% Triton X-100 in PBS. Following three washes with 1 × PBS, cells were treated with blocking buffer (1-mg/ml BSA in 1 × PBS) at room temperature for 30 min and were incubated with primary antibodies (1:100) at room temperature for 2 h. After three washes with wash buffer (0.1% NP-40 in 1 × PBS), cells were incubated with secondary antibodies (1:200) in blocking buffer at room temperature for 1 h and washed three times with wash buffer. For double staining, two antibodies were used together. Images were taken using a confocal microscope (Olympus) and were analyzed with Fluoview Ver. 2.0b Viewer (Olympus).

Cells were grown in glass-bottomed culture dishes (MatTek) for 16 h and were transfected or not transfected with 50-nM fluorescent-labeled ASOs for 4–8 h. For free uptake, cells were incubated with 2-μM fluorescent-labeled ASOs for 16–24 h. Immunofluorescence staining was performed as described (22 (link)), with minor revisions. Briefly, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and were permeabilized for 5 min using 0.1% Triton X-100 in PBS. Following three washes with PBS, cells were treated with blocking buffer (1-mg/ml BSA in PBS) at room temperature for 30 min and were incubated with primary antibodies (1:100–1:200) in blocking buffer at room temperature for 2–4 h, or at 4°C overnight. After three 5-min washes with wash buffer (0.1% NP-40 in PBS), cells were incubated with secondary antibodies (1:200) in blocking buffer at room temperature for 1–2 h and washed three times (5 min each) with wash buffer. For double staining, two antibodies were used together. Anti-fade reagent containing DAPI (Life Technologies) was added, and slides were covered with cover slips. Images were taken using a confocal microscope (Olympus, FV-1000) and were analyzed with Fluoview Ver. 2.0b Viewer (Olympus).