Biotinylated anti cd45 antibodies

For each mouse analyzed, the right lung was extracted and fixed with formalin. Lungs were paraffin embedded and tissue processed for hematoxylin-eosin staining as described (16 (link)).
Immunofluorescence assay for detection of the SARS-CoV-2 N antigen and leukocyte infiltration were performed using rabbit anti-N (Rockland chemicals, Limerick, PA, USA)/anti-rabbit IgG Alexa 488 (Jackson Immuno Research lab, West Grove, PA, USA) and biotinylated anti-CD45 antibodies (BD Bioscience, Franklin Lakes, NJ, USA)/CY3 streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) was as described (17 (link)).

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.