RNAscope Multiplex V2 manual assay from ACDbio was performed on paraffin-embedded 5 μm slices of lung tissue using mild digest times according to manufacturer instructions as we have described. Probes used were RNAscope Probe-V-nCoV2019-S-C3 (catalog number 848561) with Akoya Bio Opal Dye 520 using the 488 nm laser line and RNAscope Probe-nCoV2019-orf1ab-sense-C2 (catalog number 859151) with Opal Dye 690 using 640 nm laser line. After RNAscope assay was complete, slides were washed in TBST (1X TBS pH 7.6 with 0.1% Tween-20) for 2 minutes with agitation twice. Slides were incubated in the dark at room temperature for 30 minutes with 10% normal goat serum in 1X TBS with 1% bovine serum albumin (BSA). The blocking solution was removed from slides via gentle flicking. Slides were then incubated in primary antibody solution containing anti-CD206 antibody (clone C-10) conjugated to AF546 (Santa Cruz Biotechnology Cat# sc-376232, RRID:AB_10989352) at 1:100 dilutions in TBS 1% BSA for 1 h at room temperature in the dark. Slides were rinsed using TBST for 5 minutes with agitation twice. Slides were rinsed in TBST buffer for 5 minutes twice. Slides were then mounted and dried overnight. Images were acquired at the Center for Advanced Microscopy at Northwestern University Feinberg School of Medicine using the Nikon W1-Spinning Disk Confocal microscope. Nucleus was added to the images using machine-based learning network trained on one patient using DAPI and brightfield images in Nikon Elements. Final images were rendered using Fiji.

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