Fitc donkey anti rabbit igg

Cells cultured on slides were fixed with 4% paraformaldehyde, treated at 4 °C with methanol for 1 min, and then blocked with 1% goat serum and 5% BSA for 1 h. The cells were stained with antibodies to CD146 (1:50 dilution), nucleostemin (1:100 dilution), or BrdU (1:100 dilution) at room temperature (RT) for 3 h. The slides were subsequently incubated with FITC-donkey anti-rabbit IgG or Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution; BioLegend, San Diego, CA) for 20 min and then counterstained with Hoechst 33258 for 6 min. The slides were rinsed with PBS with Triton X100 (0.5%) three times, mounted with FluorSave™ reagent (Calbiochem) and viewed with a Zeiss epifluorescence microscope.

AD100-gp96-Ig cytospins were fixed in pure cold acetone (VWR chemicals, BDH^®, Catalog #BDH1101) for 10 min followed by three washes of 5 min each with phosphate-buffered saline (PBS). The slides were left in blocking media [5% bovine serum albumin (BSA) in PBS] at room temperature for 2 h. Rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam ab272504) and Donkey anti-rabbit IgG FITC, (BioLegend Cat# 406403) fluorescent antibody—were added in 1/50 and 1/100 dilutions of the antibodies combined in 5% BSA in PBS and/or rabbit isotype control (Abcam Ab172730 diluted 1/50), and incubated overnight at 4° C in a dark moisture chamber. The next day, slides were washed 3 times for 5 min with PBS and mounted with Prolong Gold antifade reagent with DAPI from Invitrogen (Catalog #36935), covered with a coverslip, and allowed to cure. The slides were then sealed with nail polish and taken to the KEYENCE microscope for examination. The following filter cubes were used: DAPI (for nuclear stain), FITC (for protein S), and images were acquired on KEYENCE microscope (BZ-X Viewer).

At 24 hr before transfection, 1x10⁶ cells of HEK293T were seeded in 6-well plate (Thermo Fisher Scientific, MA, USA). The cells with approximately 80–90% confluency were separately transfected with individual recombinant plasmid constructs (pCMVkan-S, -S1 and -S2) using lipofectamine 3000™ (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. Briefly, 2.5 μg of plasmid and 3.75 μL lipofectamine 3000™ were separately diluted in 125 μL and 250 μL serum-free Opti-MEM™ medium (Gibco), respectively. P3000™, a transfection enhancer reagent provided in lipofectamine 3000™ kit, was also added into diluted plasmid tube to a final concentration of 2 μL/μg DNA. The diluted DNA was mixed with the diluted lipofectamine 3000™ at 1:1 ratio (v/v) and incubated for 15 min at room temperature. The plasmid-lipofectamine 3000™ complex was then added onto cells. At 24 hr post-transfection, cells were fixed, permeabilized with ice-cold acetone and stained with 1:200 dilution of anti-S1 or anti-S2 polyclonal antibodies (Sino Biological, Beijing China). Donkey-anti-rabbit IgG-FITC, 1:5000 (BioLegend, USA), was used as a secondary antibody following anti-S1, or anti-S2 staining. Cell nuclei were counter stained with 4, 6-diamino-2-phenylindole hydrochloride (DAPI) (Sigma-Aldrich, USA). Stained cells were visualized under fluorescence microscope (Olympus, Japan).