Ti2 e widefield microscope

Infected hamster lungs were fixed for 24 h in 10% formalin. The fixed samples were washed with 70% ethanol and embedded in paraffin by TP1020 Leica semi-enclosed benchtop tissue processor (Leica Biosystems, Buffalo Grove, IL, USA) and sectioned with microtome (Thermo Fisher Scientific). Sectioned samples were dewaxed by serially diluted xylene, ethanol, and double-distilled water in sequence. To retrieve the antigens, the sectioned samples were boiled with antigen unmasking solution (H-3300, Vector Laboratories) at 85 °C for 90 s, followed by Sudan black B and 1% BSA blocking for 30 min, respectively. The in-house rabbit anti-SARS-CoV-2-N immune serum or in-house rabbit anti-SARS-CoV immune serum was used as the primary antibodies to stain the N protein of SARS-CoV-2 or SARS-CoV accordingly by incubation at 4 °C overnight. The secondary antibody, goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (A-11008), was purchased from Thermo Fisher Scientific. The anti-fade mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA) was used for mounting and DAPI staining. Images were acquired by using Nikon Ti2-E Widefield Microscope (Japan, Tokyo).

Alveolar BC were seeded into two wells of a 24-well plate. When the cells reached about 70% confluence, one of the wells was stained with live cell stain NucBlue, imaged at 4 × using a Nikon Ti2-E widefield microscope and the cells counted by using the general analysis NIS software version 4.2.1 (time point 0). The same procedure was repeated with the second well after 48 h.