Prolong glass antifade mountant

Stably transfected cells were used for imaging and cells were seeded at a confluency of ~ 30% on 10-mm Poly-L-Lysine coated coverslips. Two days post-seeding the media was removed, cells were washed once with PBS and then fixated for 20 min at room temperature in 4% paraformaldehyde in PBS. Cells were permeabilized for 5 min using 5% (w/v) bovine serum albumin (BSA) supplemented with 0.1% (v/v) Triton X-100 and then blocked by one-hour treatment with 5% (w/v) BSA at room temperature. For detection of the proteins, cells were incubated with primary antibodies diluted in 5% (w/v) BSA for 2 h at room temperature, followed by secondary antibodies diluted 1:400 in 5% (w/v) BSA for one hour at room temperature. Finally, cells were incubated for 5 min with 1:10,000 40,6-Diamidino-2-phenylindol (DAPI, 5 μg/ml) solution at room temperature to visualize the cell nuclei. Coverslips were loaded onto glass plates using ProLong Glass Antifade Mountant (Thermo Fisher, Waltham, USA). Pictures were taken using a LSM 800 Zeiss Observer Z.1 confocal microscope with a 60 × oil lens and 2 × optical zoom. The intensity of the pictures was increased for visualization purposes with the Zen lite 2011 program.

Thin sections of lung tissue were subjected to deparaffinization, rehydration and antigen recovery. Endogenous peroxidases were then blocked, followed by tissue permeabilization using 0.4% Triton X-100 in PBS. Nonspecific binding was blocked, and the thin sections were washed twice with PBS containing 0.05% Tween 20 and incubated with anti-SARS-CoV-2 spike glycoprotein rabbit antibody (Abcam, ab272504, 1:100) for one hour at room temperature. The thin sections were then washed with PBS containing 0.05% Tween 20 twice and incubated with the Alexa Fluor 647-conjugated anti-rabbit antibody (Thermo Fisher Scientific; A21244, 1:100) for 30 min in a dark chamber at room temperature. After washing, anti-horse IgG FITC (Sigma-Aldrich; F-7759, 1:100) was added and incubated for one hour at room temperature. The thin sections were washed, and the slides were mounted using Hoechst (Thermo Fisher Scientific; 62249, 1:1000) and ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific; P36980). The sections were analysed under a confocal microscope (Leica TSC SP8 DSL Hyvolution). The images were assembled and analysed in 3D using Imaris Viewer software.

DRGs were plated on 8-well chamber slides (LabTek) coated with poly-D-lysine and cultured for 5 days. After use in an assay, cultures were washed once with warm PBS then fixed for 15 min in 4% formaldehyde. Cultures were washed three times with wash buffer (1% BSA in PBS, same for all subsequent washes). Afterward, cells were permeabilized with 0.5% TritonX100 (in PBS) for 5 min. To remove the detergent, samples were washed three times. Samples were blocked with addition of 8% goat serum (Sigma, diluted in wash buffer) for 1 h at ambient temperature (22–24 °C). After blocking, the serum was aspirated and primary antibodies diluted in 8% goat serum were added onto the samples and allowed to incubate overnight at 4 °C. DRG neurons were labeled with antibodies against RCK (MBL, 1:1000), peripherin (Novus, 1:1000), and eEF2K (Invitrogen, 1:500). U2-OS cultures were labeled with antibodies against RCK (1:500, SCBT), and phalloidin-TRITC (1:200). Samples were washed three times before adding secondary antibodies (all 1:1000) diluted in 8% goat serum. After for 1 h, samples were washed three times and nuclei were stained with DAPI (0.1 ng/ml, Sigma) for 10 min. The chambers were removed from the slides, and coverslips were mounted using ProLong Glass antifade mountant (ThermoFisher). Slides were fixed using clear nail polish.

HEK-293T cells were plated on glass coverslips coated with poly-D-lysine (Sigma) and cultured for 24h before transfection. Transfection was done as described above. Cells were rinsed with ice-cold PBS and fixed in 4% formaldehyde/PBS for 10min at room temperature. Cells were blocked for 1h at room temperature in 10% normal goat serum (Thermofisher) and then sequentially incubated in primary antibodies (overnight at 4C with rabbit α-GFP (Thermofisher, A11122) or mouse α-FLAG (Thermofisher, MA191878)). Labeled cells were washed three times with ice-cold PBS and incubated in secondary antibody for 1h at room temperature and DAPI (Sigma) for 1min at room temperature. Cells were mounted onto glass slides in ProLong Glass Anti-fade mountant (Thermofisher). Fluorescently conjugated secondary antibodies used were Alexa Fluor goat anti-mouse 568 and Alexa Fluor goat anti-rabbit 488 (Thermofisher). Cells were imaged on a Zeiss AxioImager at 40X magnification. Figures show representative imaged from at least three independent experiments for each set of transfected conditions.

On the day before treatment, cells were grown on glass coverslips (Marienfeld). After treatment, cells were fixed with 4% formaldehyde-PBS for 30 min on ice and quenched with 50 mM NH₄Cl for 15 min. For immunofluorescence labeling, cells were then permeabilized with 0.2% Triton X-100-PBS for 15 min, or 50 µg/ml digitonin (Roth, #4005) for 5 min according to the antibody manufacturers. Samples were blocked with 3% BSA (Roth, #8076)-PBS for 30 min and incubated with primary antibodies for 1–2 h. After washing and 30 min of secondary antibody incubation, samples were again washed three times with PBS. Cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, #P36980), including DAPI. Imaging was performed with a Zeiss Axio Observer 7 fluorescence microscope (Zeiss, Köln, Germany) with a Plan Apochromat 40x/1.4 oil objective (Zeiss, Köln, Germany). Quantification of images was performed with ImageJ. For that, signals and nuclei were counted per image and a signal-to-nuclei ratio was calculated. Macros for the quantifications are provided in Supplementary Methods.