Alexa fluor 594 conjugated donkey anti mouse igg

Intracellular localization of the exogenous WT or Δ8 human SIGIRR was examined by IF staining. The cells, plated on glass-bottom dishes, were washed twice with a phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) (Nacalai Tesque, Inc., Kyoto, Japan) for 30 min at room temperature (RT) and were then rinsed with PBS twice. Cells were non-permeabilized or permeabilized with 0.5% TritonX-100 in PBS for 20 min and blocked with 3% donkey serum for 1 h at RT. Cells were incubated for 1 h at RT with primary antibodies against a FLAG tag (1:250; Wako, Osaka, Japan), Myc tag (1:250; Wako, Japan), or PDI (1:250; Proteintech, Rosemont, IL, USA) diluted in 1% donkey serum in PBS. Then, the cells were washed three times with PBS, and an Alexa Fluor^® 594-conjugated donkey anti-mouse IgG (1:500; Abcam, Cambridge, UK) and Alexa Fluor^® 488-conjugated donkey anti-rabbit IgG (1:500; Thermo Scientific Inc.) were added to the cells, which were then incubated for 45 min at RT with shielding. After washing the cells three times with PBS, the cells were mounted with a Vectashield^® antifade mounting medium with a DAPI (Vector Laboratories, Burlingame, CA, USA). The images were visualized and captured with a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan) using the appropriate filters.