Metamorph image acquisition software

Spatial IHC and sm-FISH images were acquired using an Axiovert 200 M inverted fluorescence microscope (Zeiss, Oberkochen, Germany) using a 20x or 63x oil-immersion objective and a Prime sCMOS camera (Photometrics, Tucson, AZ, USA) controlled by Metamorph image acquisition software (Molecular Devices, San Jose, CA, USA). To enumerate the various cell types in the lungs that express the tested target molecules (CD3, CD11B, CD56, CD68, IBA-1, GR, GSTM3 and F4/80), image analysis was conducted using ImageJ software ver. 1.46 (National Institutes of Health, Bethesda, MD, USA). The number of cells positive for a specific host cell marker or signal intensities from various channels in 10–15 fields of fluorescently stained tissue sections were measured. Pooled average field intensity measured from at least 5 fields per slide containing minimum 100 cells per field was used for calculations.

Cells were fixed in 4% PFA in phosphate-buffered saline (PBS) at room temperature (RT) for 30 min and blocked with 3% bovine serum albumin in PBS (3% B-PBS). Cells were incubated with primary antibodies in 3% B-PBS at 4 °C overnight. Secondary antibodies conjugated to Alexa Fluor 594 (Thermofisher) or Alexa Fluor 488 (Thermofisher) in 3% B-PBS were added and incubated at RT for 1 hr, followed by several washes with PBS. The staining with transferrin-594 conjugate (Thermofisher) was performed following the manufacturer’s protocol. For HLC cultures grown on transwell filters, the filter was removed from the hanging insert and submerged, cell side up, in PBS using a slice anchor (Warner Instruments). Cultures were imaged on an Upright BX61WI microscope (Olympus) with a UMPlan FL 60 × 1.0 NA water dipping objective (Olympus) or UMPlan FL ×10, 0.3 NA water dipping objective and an Orca Flash 4.0 digital CMOS camera (Hamamatsu) using MetaMorph image acquisition software (Molecular Devices). Deconvolution was performed using the standard adaptive point spread settings in Autoquant (Media Cybernetics). Image analysis was conducted using Fiji.

The formalin-fixed, paraffin-embedded tissue blocks were sliced into 5-micron sections and processed following standard procedures [18 (link)]. The sections were stained with rabbit anti-hamster ACE2 antibody (Product no. HPA000288, Millipore Sigma, Burlington, MA, USA) followed by Alexa-488 labeled anti-rabbit secondary antibodies (Cat no. ab150077, Abcam, MA, USA), as described previously [19 (link)]. For the mRNA-FISH technique, labeled smRNA-FISH probes (Biosearch Technologies, Dexter, MI, USA) corresponding to specific host genes were added to the top of the sections and processed as described previously [19 (link)]. Images were captured using Axiovert 200 M inverted fluorescence microscope (Zeiss, Oberkochen, Germany) using 20× or 63× oil-immersion objective with Prime sCMOS camera (Photometrics, Tucson, AZ, USA) by Metamorph image acquisition software (Molecular Devices, San Jose, CA, USA), ImageJ software was used for analyses. The number of cells positive for a marker was normalized to the total number of cells in each field (3–5 fields containing at least 100 cells were analyzed per sample). Further, GraphPad Prism-8 (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis of data. p-values < 0.05 were considered statistically significant.