Goat anti chicken igy h l alexa fluor 488

To examine the SARS-CoV-2 RBD and S1 subunit proteins expression, Vero-E6 cells were infected with the recombinant rLS1-HN-RBD, rLS1-S1-F or rLS1 viruses at a multiplicity of infection (MOI) of 0.5. After 48 hours post-infection (hpi), the cells were fixed with 4% paraformaldehyde for 25 minutes (min), and then the monolayer was washed three times with Dulbecco’s phosphate-buffered saline (DPBS) and permeabilized with Triton 0.1% X-100 for 15 min at room temperature (RT). After washing with the cells with DPBS, the monolayer was incubated with the rabbit polyclonal antibody specific to SARS-CoV-2 RBD protein (1:200) (Sino Biological, Beijing, China), and a chicken antiserum specific to Newcastle disease virus (1:200) (Charles River, Norwich, CT, USA) for 1.5 h at RT. Afterwards, the monolayer was incubated with Donkey Anti-Rabbit IgG H&L-Alexa Fluor® 594 (1:250) and Goat Anti- Chicken IgY H&L-Alexa Fluor® 488 (1:1000) (Abcam, Cambridge, MA, USA) for 60 min at RT. Finally, the cells were developed with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and observed using an ObserverA1 fluorescence microscope (Carl Zeiss, Germany). Digital images were taken at 400× magnification and processed with the AxioCam MRc5 camera (Carl Zeiss, Germany).

HEK293 cells and Cos-7 cells were grown on 20 × 20 mm coverslips placed in 6-well plates, then transfected with 1.2 µg FLAG-hCaSR and EGFP-CaSR DNA, respectively, and allowed to grow for 48 h prior to immunostaining. Cells were washed with ice cold PBS and fixed with 3.7% formaldehyde for 15 min at room temperature, followed by wash with PBS three times. Cells were permeabilized using 0.2% Triton X in PBS for 10 min at room temperature. HEK293 cells were incubated with mouse anti-FLAG monoclonal antibody at 1:1000 dilution and goat anti-mouse IgG (H + L) Alexa Fluor 488 secondary antibody (A32723, Invitrogen) for 1 h each at room temperature to stain FLAG-CaSR. Cos7 cells were incubated with anti-GFP antibody at 1:2000, anti-calreticulin antibody at 1:200, and anti-VAPA antibody at 1:125 or anti-GRP78 antibody at 1:100 in PBS with 3% BSA at room temperature for 1 h. The cos-7 cells were subsequently washed with PBS and stained with secondary antibodies goat anti-chicken IgY H&L Alexa Fluor 488 (ab150173, Abcam), donkey anti mouse IgG (H + L) Alexa fluor 647 (A31571, Invitrogen) and goat anti-mouse IgG (H + L) Alexa Fluor 555 (A-21422, Thermo Fisher Scientific), respectively for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole.