Alexa fluor 594 goat anti mouse igg h l

To demonstrate AChR clustering, the cells were visualised on an Olympus IX71 fluorescence microscope. Between 2 or 3 days after transfection, cells were incubated with mAb C7 for 1 hour at RT, which binds the extracellular domain of the AChR δ-subunit,15 (link) diluted 1:1000 in staining medium (DMEM containing 20 mM Hepes and 1% bovine serum albumin (BSA)), before being washed three times with staining medium. Cells were fixed with 3% paraformaldehyde at RT for 10 min, washed three times with phosphate-buffered saline (PBS) and incubated with secondary antibody Alexa Fluor 594 goat anti-mouse IgG (H+L) (catalogue no. A11005; Invitrogen; RRID: AB141372) diluted 1:750 in staining medium. Cells were washed three times in PBS and mounted in fluorescence mounting medium (Dako Cytomation, Glostrup, Denmark). Images were captured using Simple PCI (Digital Pixel).

The protein expression levels of TGEV N protein in candidate gene KO cells and control cells were determined via an immunofluorescence assay. Cells grown on the 12-well cell culture plates were first infected with TGEV at different MOIs. At 24 hpi, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and then permeabilized at room temperature for 10 min with cold 0.3% TritonX-100 in phosphate buffered saline (PBS). Cells were incubated with TGEV N antibody (A rabbit anti-TGEV N protein polyclonal antibody was prepared in our laboratory) or anti-dsRNA antibody (SCICONS, #10010200, 1:1,000) at 4°C overnight, and the primary antibodies were recognized by Alexa Fluor 594 Goat anti-Mouse IgG (H+L) (Invitrogen, #A-11005, 1:1,000), Alexa Fluor 594 Anti-Rabbit IgG (H + L) (Invitrogen, #A-11012, 1:1,000), or Alexa Fluor Plus 647 Goat anti-Mouse IgG (H+L) (Invitrogen, #A32728, 1:1,000). Cell nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma, #D9542) at room temperature for 1 min in the dark. Cells were observed and imaged with a fluorescence microscope (Thermo Fisher Scientific EVOS FL Auto). The proportion of positive cells were calculated using Image J software (three independent wells were imaged, and one random field of view per well was captured for each experimental phase).

Macrophages were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton™ X-100 for 10 min, and blocked with 1% BSA for 1 h. The cells were then incubated with primary antibodies: rabbit anti-human CD86 (ab53004, Abcam, Cambridge, UK) at 1:500 dilution or mouse anti-human CD206 (170710, Bio-Rad, USA) at 1:200 dilution overnight at 4°C. The double-stranded RNA (dsRNA) was stained with anti-dsRNA monoclonal antibody (English & Scientific Consulting Kft.; K1-1301) at 1:2,000 dilution. The secondary antibodies Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A-11034, Invitrogen, USA) or Alexa Fluor 594 goat anti-mouse IgG (H + L) (A11032, Invitrogen, USA) were used at 1:1,000 dilution for 1 h. 4′,6-Diamidino-2-Phenylindole (DAPI) was used to stain the nucleus at a concentration of 100 ng/ml.