Axioscan 7 instrument

Formalin-fixed and paraffin-embedded tissues sectioned to 4 μm were used for histological evaluation of liver tumors in a mouse model. Hematoxylin and eosin (H&E) staining was performed for each sample. For IHC, tissue slides were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions (100%, 95%, and 70%). Subsequently, the slides were subjected to antigen retrieval by microwaving in a citric acid solution for 15 min. The primary antibodies anti-hepatocyte (Abcam, Cat#ab75677), anti-Epcam (Abcam, Cat#ab213500), anti-CD3 (Abcam, Cat#ab16669), anti-Ly6G (Servicebio, Cat#GB11229), anti-CD8 (Abcam, Cat#ab217344), anti-F4/80 (CST, Cat#70076), anti-Asma (Servicebio, Cat#BM0002), and anti-CD31 (Servicebio, Cat#GB113151) were used. Subsequently, the slides were incubated with secondary antibodies (1:1, 100 μL for each slide; HRP-anti-rabbit IgG, ZSGB, Cat#PV-6001, or HRP-anti-mouse IgG, ZSGB, Cat#PV-6002) for 10 min at room temperature. Multispectral images were scanned with a ZEISS AXIOSCAN 7 instrument. Cells of interest were quantified in Halo v3.4 (Indica Labs) or QuPath v0.2.0. Each section was evaluated by 2 or 3 experienced pathologists.

For each group analyzed (N = 5 mice/group), the right lung lobe was extracted, fixed in formalin and paraffin embedded as described^{29 (link)}. Lung sections were stained with Carstairs staining for histological analysis. Prior to conduct immunofluorescence assay, lung sections were deparaffined, hydrated then heat-induced epitope retrieval 16 h at 60 °C with Diva Decloaker solution (Biocare Medical, Pacheco, CA, USA). Immunostainings were preformed to detect SARS-CoV-2 N antigen and leukocyte infiltration using 20 µg/mL rabbit anti-N (Rockland chemicals, Limerick, PA, USA)/anti-rabbit IgG Alexa 488 (Jackson Immuno Research lab, West Grove, PA, USA) and 10 µg/mL biotinylated anti-CD45 antibodies (BD Bioscience, Franklin Lakes, NJ, USA)/anti Rat IgG Alexa Plus 647 (Thermo Fisher Scientific, Waltham, MA, USA). Slide were imaged using Axioscan 7 instrument (Carl Zeiss Microscopy, New York, USA) then black and white adjustment were performed with Zen lite 3.7 (Carl Zeiss Microscopy). Quantification of positive area signal for N and CD45 staining were performed using Fiji (ImageJ) threshold analyse tools.