4 6 diamidin 2 phenylindol

Microscopical pictures of cell–cell fusion were taken with an Axiovert 200 fluorescence microscope with a Colibri 7 light source, Axiocam 512 mono, and data analysis and controlling were performed using Zen software (Zeiss, Jena, Germany).
Microscopical pictures of SARS-CoV-2 S mediated cell–cell fusion were obtained by fixing cells with 3.7% formaldehyde, permeabilization by Triton X-100 0.5% in PBS and staining with human anti-S polyclonal serum (1:100), goat-anti-human F(ab)₂ biotin (1:500; Dianova), streptavidin-FITC (1:500; Life technologies,) and 4′,6-diamidin-2-phenylindol (1:1000; Sigma-Aldrich).
Following methanol fixation, the Giemsa-staining of cells was performed using Giemsa staining solution (Sigma-Aldrich/Merck) according to the manufacturer’s instructions.

IF was performed as reported previously.^{23 (link)} Incubation with primary antibodies CD271-PE (Miltenyi, Bergisch Gladbach, Germany, clone ME20.4-1.H4, mouse IgG1, 1:100), γH2AX (1:250, Cell Signaling Technology), NFκB/p65 and p53 (Cell Signaling Technology, 1:100) or TRITC-labeled phalloidin (1:1000, Sigma-Aldrich) diluted in blocking buffer was done overnight at 4 °C. Second day, cells were washed 3x with phosphate-buffered saline, and incubated with secondary antibodies AlexaFluor488/555 or 594 (1:500) recognizing either rabbit or mouse-produced antibodies and 4′,6-diamidin-2-phenylindol (Sigma-Aldrich, 1:500) for 1 h at room temperature. Washed cells were covered with 500 μl phosphate-buffered saline and used for microscopy. IF pictures were recorded with Zeiss Axiovert40CFL with accompanied Illuminator HPX120C and software AxioVision Rel. 4.8 (all Carl Zeiss AG, Oberkochen, Germany).

Cells plated on PDL-coated glass coverslips were blocked with 2% BSA, 0.5% Triton-X (in PBS) for 1 hr prior to immunostaining. Primary antibodies (chicken alpha-GFP, Aves Labs: GFP-1010 and rabbit alpha-Ki67, abcam: ab92742) were applied in blocking solution overnight at 4°C. Fluorescent secondary antibodies were applied in blocking solution for 1 hr at room temperature. DAPI (4’,6-diamidin-2-phenylindol, Sigma) was used to visualize nuclei. Stained cells were mounted in Aqua Polymount (Polysciences). All secondary antibodies were purchased from Life Technologies. Representative high-quality images were taken using an Olympus FV1000 confocal laser‐scanning microscope using 20×/0.85 NA water immersion objective. Images used for quantification were taken using an epifluorescence microscope (Zeiss, Axio ImagerM2) equipped with a 20×/0.8 NA and 63×/1.25 NA oil immersion objectives. Postimage processing with regard to brightness and contrast was carried out where appropriate to improve visualization, in a pairwise manner.