Nucleolus bright red

Cells were plated in 8-well chamber slides (Matsunami, Osaka, Japan) coated with rat collagen I (Corning, Corning, NY). Infected or transfected cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; Nacalai Tesque, Japan) for 10 min and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. The cells were blocked with Blocking One solution (Nacalai Tesque) for 30 min, followed by incubation with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. For nuclei and rRNA staining, the cells were treated with Hoechst 33342 (Thermo Fisher Scientific) and Nucleolus Bright Red (Dojindo, Japan), respectively, for 10 min. Section images were recorded using DeltaVision Elite (GE Healthcare) with a 60× oil objective and then deconvolved and projected using the Quick Projection tool by softWoRx (GE Healthcare).

Transfected cells were placed on a 35-mm glass-bottom dish (IWAKI) coated with poly-L-Lys. Before observation, the media was substituted with D-MEM (HEPES, no Phenol Red). Samples were observed by confocal laser fluorescence microscopy (CLSM; LSM780, Zeiss) with ZEN2.3 (black) software. We used a 405 nm laser diode for Hoechst 33342 (Dojindo) and DAPI, a 488 nm argon laser for sfGFP, mAG, and AG, a 561 nm diode-pumped solid-state laser for mCherry, DsRed2, and Nucleolus Bright Red (Dojindo), and a 633 nm He–Ne laser for Alexa fluor 633.
For imaging the proteins in PBS, samples were placed on a glass-bottom 96-well plate and observed by fluorescent microscopy (ECLIPSE TI-FL, Nikon or AxioObserverZ1, Zeiss, ZEN2.6 (blue) software).