Collagen coating glass bottom culture dishes

Cells seeded on collagen coating glass-bottom culture dishes (Matsunami Glass Ind., Ltd.) were transfected with 250 nM mPCS1f or BRO (mPCS1f/PNA(C8)) and incubated for 48 h at 37 °C and 5% CO₂. After rinsing thrice with 1× PBS, cells were fixed at room temperature with 4% formaldehyde for 10 min and permeabilized with 0.1% NP-40 in 1× PBS for 10 min. Fixed cells were blocked in Blocking One Histo (Nacalai Tesque) at room temperature for 30 min and then incubated in 20× diluted Blocking One Histo (Nacalai Tesque) with primary antibodies at room temperature for 2 h. After washing thrice with PBS-T (5 min per wash), cells were incubated in 20× diluted Blocking One Histo with secondary antibodies at room temperature for 1 h. Finally, cells were washed thrice with PBS-T (5 min per wash) and nuclei were stained with Hoechst 33258 (Dojindo) for 10 min. The cells were mounted using Fluoromount (Diagnostic Biosystems). Confocal images were generated using confocal laser scanning microscopy (LSM710 microscope, Carl Zeiss Co., Ltd.) and analyzed by ZEN software 3.5. 093. 00001 (blue edition). The 3D structure was rendered in PyMol (Version 2.4.1).

Cells seeded on collagen coating glass-bottom culture dishes (Matsunami Glass Ind., Ltd.) were transfected with 250 nM mPCS1f or BRO (mPCS1f/PNA(C8)) and incubated for 48 h at 37 °C and 5% CO₂. After rinsing thrice with 1× PBS, cells were fixed at room temperature with 4% formaldehyde for 10 min and permeabilized with 0.1% NP-40 in 1× PBS for 10 min. Fixed cells were blocked in Blocking One Histo (Nacalai Tesque) at room temperature for 30 min and then incubated in 20× diluted Blocking One Histo (Nacalai Tesque) with primary antibodies at room temperature for 2 h. After washing thrice with PBS-T (5 min per wash), cells were incubated in 20× diluted Blocking One Histo with secondary antibodies at room temperature for 1 h. Finally, cells were washed thrice with PBS-T (5 min per wash) and nuclei were stained with Hoechst 33258 (Dojindo) for 10 min. The cells were mounted using Fluoromount (Diagnostic Biosystems). Confocal images were generated using confocal laser scanning microscopy (LSM710 microscope, Carl Zeiss Co., Ltd.) and analyzed by ZEN software 3.5. 093. 00001 (blue edition). The 3D structure was rendered in PyMol (Version 2.4.1).