A1 high sensitivity confocal microscope

Organoids were grown as previously described (75 (link)) and infected with SARS-Cov-2 (non-tagged or GFP-tagged virus) as described above. Organoids were fixed 2 days post-infection with ice-cold 4% paraformaldehyde (Electron Microscopy Sciences) for 45 min at 4°C. Organoids were either cryosectioned and processed for immunofluorescence and imaging as described (75 (link)) or left as whole organoids and processed for high-resolution immunofluorescence and imaging per published protocols (79 (link), 80 (link)). Briefly, fixed organoids were blocked with ice-cold organoid washing buffer (OWB): 0.2% BSA + 0.1% Triton X-100 + 0.02% SDS in PBS for 30 min at 4°C and incubated with the primary antibodies of interest suspended in OWB overnight at 4 °C with gentle horizontal shaking, then with appropriate secondary antibodies overnight at 4 °C. Organoids were stained with DAPI for 10 min, washed, cleared with FUnGI (50% (v/v) glycerol, 9.4% (v/v) dH2O, 10.6 mM tris base, 1.1 mM EDTA, 2.5 M fructose and 2.5 M urea) prepared as previously described(80 (link)) and mounted in FUnGI. Sample images were captured using a Nikon A1 High Sensitivity Confocal microscope and processed using Nikon Elements software.

PLO/lam-coated coverslips with SCA3 NPCs were washed with PBS, fixed with 4% paraformaldehyde/ PBS (-Mg²⁺, -Ca²⁺, HyClone) for 15 min, washed three times with PBS and stored at 4°C until further processing. Cells were permeabilized with 0.5% Triton X-100/ PBS for 20 min, washed with 0.1% Tween-20/ PBS (PBS-T), blocked in 5% goat serum/ PBS for one hour, and incubated overnight at 4°C with primary antibodies diluted in 5% goat serum/ PBS: rabbit anti-PAX6 (1:250, 60433S, Cell Signaling), rabbit anti-SOX1 (1:1000, 4194S, Cell Signaling), mouse anti-NESTIN (1:250, 33475S, Cell Signaling), rabbit anti-MJD (1:1000), and mouse anti-ATXN3 (1H9) (1:250, MAB5360, Millipore). Cells were washed with PBS-T, incubated with corresponding secondary antibodies goat anti-rabbit and anti-mouse conjugated with Alexa Fluor 488 or 568 (1:1000, Invitrogen) diluted in 5% goat serum/ PBS for 1h, incubated with DAPI for 10 min, and washed with PBS-T. Immunostained coverslips were then mounted in slides using Prolong Gold medium (Invitrogen) and saved at 4°C until imaged on a Nikon A1 high sensitivity confocal microscope.