Axiocam 512 mono

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.

Analysis of UTX-TagGFP2 localization was performed in stably transduced UCCs. As previously described [33 (link)], after fixation with 4% formaldehyde, cells were permeabilized using 0.2% Triton X100 in PBS for 10 min at RT, blocked with 1% BSA in PBS, for 30 min at RT and subsequently incubated for 1 h at RT with 14 nM Rhodamine Phalloidin in blocking solution. Following counter-staining of nuclei with 1 µg/mL DAPI (4´,6-diamidino-2-phenylindole) cells were mounted with fluorescence mounting medium (DAKO, Glostrup, Denmark). Imaging was performed using ZEISS Axio Observer.Z1 / 7; Plan-Apochromat 40x/1.4 Oil DIC (UV) VIS-IR M27; 90 HE DAPI/ GFP/ Cy3 /Cy5; LED-module "wavelength" nm (Colibri 7); Axiocam 512 mono (ZEISS, Jena, Germany)
Higher resolution images were obtained using an inverted confocal FluoView1000 laser scanning microscope with a 60xW UPLSAPO objective NA1.2 in a sequential DAPI-GFP-Phalloidin scanning mode (Olympus, Hamburg, Germany). Resolution was 1024 × 1024.

Cells were fixed and permeabilized using a solution of 1% paraformaldehyde and 0.02% Triton X-100 in PBS. All antibodies were diluted in the blocking solution (1% BSA, 0.1% saponine, 0.1% NaN₃, dissolved in PBS). Used antibodies were: KRT5 (Abcam, Cambridge, UK, ab53121, at a 1:250 dilution) and KRT14 (Abcam, ab7800, 1:250) with secondary antibodies goat-anti-mouse IgG Alexa Fluor 633 (ThermoFischer Scientific, A-21052) and goat-anti-rabbit IgG Alexa Fluor 488 (ThermoFischer Scientific, A-11008). Phalloidin (Acti-stain 535 Phalloidin, tebu-bio, PHDR1, 1:1,000) was used to stain actin filaments and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Roche, Mannheim, Germany, 10236276001) to stain DNA. Cover slips were mounted with Dako Fluorescence Mounting Medium (Dako, S3023). Detection was performed using ZEISS Axio Observer.Z1/7; Plan-Apochromat 40× /1.4 Oil DIC (UV) VIS-IR M27; 90 HE DAPI/ GFP/ Cy3 /Cy5; LED-module "wavelength" nm (Colibri 7); ZEISS Axiocam 512 mono.
Pictures were analyzed with ImageJ (version 1.52b) and a specific approach-based macro. The core adjustments were: DAPI-Channel (threshold: "Default"; "Watershed"); KRT14-Channel (threshold: "Huang"; "Watershed"). Regions of interest were counted by using "Extended Particle Analyzer" with a pixel ratio of 1:100,000.