Ci s microscope

After the mice of each group were sacrificed, the proximal colon was collected immediately, fixed with 10% formalin, embedded in paraffin, sectioned to a thickness of 5 mm, deparaffinized and submitted to hematoxylin and eosin (H&E, Sigma-Aldrich, Shanghai, China) staining. Immunohistochemistry was performed using a previously described method (Huang et al., 2019 (link)). Briefly, 3 μm sections were deparaffinized in xylene and rehydrated in graded alcohol. After quenching endogenous peroxidase activity and blocking non-specific binding, the sections were incubated with a rabbit monoclonal antibody [EPR22566-344] against c-Kit (Abcam, ab256345) overnight at 4°C and then incubated with the secondary antibody goat anti-rabbit IgG (H + L) HRP (ab0101, Abways) at room temperature for 30 min. Finally, the slides were incubated with reagents from the Avidin-Biotin Complex Kit (Vector Laboratories, Inc., Burlingame, USA) and a 3,3′-diaminobenzidine kit (Tiangen, China) according to the manufacturer’s instructions. Images were captured with a Nikon Ci-S microscope and Nikon DS-U3 imaging system. The proportion of c-Kit-positive cells in the colonic muscle layer was determined using an image analyzer (Image-Pro Plus 6.0, Media Cybernetics, Inc., Rockville, USA).

For hematoxylin and eosin (H&E) histopathology evaluation, lungs were rapidly collected, and fixed in 4% formaldehyde for 48 h, followed by paraffin embedding. Serial sections with 4 µm thickness were prepared and selected sections (5–18 sections per animal) were stained with H&E for light microscopy examination. Images were captured using NIKON CI-S microscope equipped with a DS-FI2 camera. The lung tissue lesions were assessed according to the extent of thickened alveolar septa, congestion and edema, infiltration of inflammatory cells, degeneration of epithelial cells in bronchiole tubes, damage of cilium and vacuolar degeneration of endothelia cells. A semiquantitative scoring system was used to evaluate objectively the SARS-CoV-2-induced histopathological lesions. The degree of involvement in above each histopathological criterion was scored as: 0 for normal, 1–3 for minimal, 4–6 for mild, 7–9 for moderate. A maximum average score of 9 could be reached for the combined lobes per animal using this scoring system.