Ix73 confocal microscope

For the detection of RBD-HR binding to NECs in vivo, 6–8 weeks, female BALB/c mice were intranasally immunized with CCD/RBD-HR (100 μg/10 μg per mouse) with or without pretreatment with NAs. Nasal turbinates were acquired, decalcified for 3 months, embedded in paraffin, and sectioned vertically with 3 mm thickness. Sections were fixed with 4% paraformaldehyde for 5–10 min at room temperature and blocked with 10% goat serum for half an hour. Without washing, the sections were stained with the first antibody (SARS-CoV-2 Spike Antibody, Rabbit PAb, 1:1000, Cat: 40589-T62, Sino Biological) overnight at 4 °C and the second antibody (Goat-anti rabbit IgG, Alexa Fluor, 1:1000, Cat: A11008, Invitrogen) for 1 h at room temperature. Sections were observed with Olympus IX73 confocal microscope with CellSens standard software 2.1 (Olympus Corporation).

Epididymal sperm cells after swim out were diluted into PBS (1:10); 400 μl of diluted sperm were loaded on poly-L-lysine (CAS 25988-63-0, Sigma Aldrich)-coated coverslips in a six-well plate and air dried for 30 min at 37°C. After removing the PBS, sperm cells were fixed in 4% PFA followed by quenching with 50 mM NH₄Cl for 15 min. Sperm cells were permeabilized by 0.02% Triton X-100 for 3 min followed by washing with PBS, incubation for 1 h at RT with Phalloidin ATTO647 (1/1,000 in 3% BSA, ab176759, Abcam), and mounting with ProLong Gold Antifade (#P36930, Life Technologies). STED micrographs were acquired using a four-channel easy3D super-resolution STED optics module (Abberior Instruments, Göttingen, Germany) coupled with an Olympus IX73 confocal microscope (Olympus, Tokyo, Japan) and equipped with an UPlanSApo × 100 (1.4 NA) objective (Olympus, Tokyo, Japan) (Mikuličić et al., 2019 (link)), available in the LIMES Imaging Facility.

For EdU labeling, dams were injected with EdU (15 mg/kg, Ribobio) that was diluted in PBS 2 h prior to embryo harvesting, collected in ice-cold PBS, and fixed overnight in 4% PFA at 4°C overnight. The tissues were then incubated with an ascending series of sucrose concentrations from 10% to 25% and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek United States Inc., Torrance, CA, United States). Cryosections (10 μm) were prepared, and immunostaining was performed as previously described (Fan et al., 2020 (link)). The cell proliferation rate was measured using the Cell-Light EdU Apollo488 in vitro Kit (Ribobio, C10310-3) and the immunofluorescence staining using the antibody recognizing phospho-Histone 3 (antibody from Millipore, 2465253). Cell apoptosis was assessed by immunofluorescence staining using the antibody recognizing cleaved-Caspase 3 (Cell Signaling Technologies, 9661). α-Actinin (Sigma, A7811) and PECAM1 (BD Biosciences, 550274) were used to stain cardiac and endothelial cells, respectively. DAPI was used to counterstain nuclei. The slides were imaged and subjected to an independent blinded analysis, using the Olympus IX73 confocal microscope and ImageJ software.