H 1200

Immunohistochemistry was performed as we previously described with slight modifications³¹ . Human lung tissue samples were collected and fixed overnight in 10% formalin. The fixed samples were embedded in paraffin by TP1020 Leica semi-enclosed benchtop tissue processor and sectioned with microtome (Thermo Fisher Scientific). Sectioned samples were prepared on glass slides and were dewaxed and dehydrated by serially diluted xylene, ethanol, and double-distilled water in sequence. Afterwards, the samples were co-heated together with antigen unmasking solution (H-3300, Vector Laboratories) at 85 °C for 90 s for antigen exposure, followed by blocking with 0.3% hydrogen peroxide for 30 min, and 1% BSA for 30 min. The in-house rabbit anti-SARS-CoV-2-N immune serum or in-house rabbit anti-SARS-CoV-N immune serum were applied as the primary antibodies and were incubated with the slides at 4 °C overnight. The signal was developed with the DAB (3,3’-diaminobenzidine) substrate kit (SK-4100, Vector Laboratories). Cell nuclei were labeled with Gill’s haematoxylin. The slides were mounted with antifade mounting medium with DAPI (H-1200, Vector Laboratories). Images were taken with the Olympus BX53 light microscope (Olympus Life Science).

Cell clusters or excised kidneys were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Science; 15,714) for 1 h at room temperature (RT) or overnight at 4 °C, respectively, before being paraffin embedded and sectioned at 10 μm. Paraffin was removed with Histoclear (Thermo Scientific; C78-2-G) and rehydrated, and antigens retrieved by 2 h 0.1 M EDTA (Ambion; AM9261) treatment in a pressure cooker (Proteogenix; 2,100 Retreiver). Samples were blocked with 0.1% Triton X-100 (VWR; EM-9400) and 5% donkey serum (Jackson Immunoresearch; 017-000-121) in PBS (staining solution) for 1 h at RT, incubated with primary antibodies diluted in staining solution overnight at 4 °C, washed for 5 min in staining solution, incubated covered with appropriate Alexa Fluor-488 or -594 secondary antibodies diluted 1:300 in staining solution 2 h at RT, washed for 5 min in staining solution, mounted with Vectashield (Vector Laboratories; H-1200) and covered with a coverslip. Images were taken with an Olympus IX51 Microscope. The primary antibodies used to assess differentiation throughout this study are given in Supplementary Table 6 (ref. 17 (link)).

Cells were grown on coverslips and after treatment, cells were fixed in (10% v/v) formaldehyde for 10 min at room temperature. After washing with phosphate buffered saline (PBS) cells were mounted on glass slides in a mountant with DAPI (Vectashield_Cat#H-1200). Nuclei were visualised using an Olympus BX61 fluorescence microscope. Apoptotic nuclei (condensed, fragmented, intensely stained) were counted and presented as a percentage of the total nuclei. At least 100 cells were counted per well, and all treatments were performed in triplicate.

After WIP1 inhibition, single cell suspension was prepared and mix with low melting point agarose at 37 °C. And spread the agarose and cell mixture on the pretreated glass slides. Treat with lysate for 2 h at 4 °C. And perform gel electrophoresis and then neutralize the slides. And after the agarose is completely dry, stain with DAPI dyes (Vectorlabs, H-1200, USA) and observe the degree of DNA damage under fluorescent microscope. The CASP Comet Analysis software. TailDNA% was used to calculate the Tail/Head DNA percent of every single cells. and the average Tail/Head DNA percent was shown as mean ± SD. More than 10 cells were analyzed.

For frozen sections, enucleated eyeballs from P0 mice and embryos were fixed in 4% paraformaldehyde for 30 min at room temperature and 30% sucrose dehydrated over night at 4°C, then embedded in OCT, frozen, and sectioned at 10 µm. Cryosections were permeabilized in 0.5% Triton X-100/PBS for 2 min, blocked in 5% donkey serum and 5% BSA in PBS for 1 h at room temperature, then incubated with primary antibodies overnight at 4°C. After rinsing, the sections were incubated with fluorescence-labeled secondary antibodies (Alexa Fluor 488, or Alexa Fluor 546, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature and counterstained with DAPI (1:1,000, H-1200, Vector Labs, Burlingame, CA, USA). Primary antibodies used for immunofluorescence were LSS (1:300, 18693-1-AP, Proteintech, Wuhan, China), p57^KIP2 (1:200, ab75974, Abcam, Cambridge, MA, USA), Pax6 (1:200, PRB-278P, BioLegend, San Diego, CA, USA), and Prox1 (1:200, 11067-2-AP, Proteintech). The images were captured with an LSM980 confocal scanning microscope (Carl Zeiss, Thornwood, NY, USA) or a TissueFAXS microscope (TissueGnostics, Austria).