Confocal Imaging of Infected Lung Tissue

For confocal immunofluorescence microscopy of the fixed, infected lungs, a lung lobe was embedded in 4% agarose, before sectioning into 250 μm sections using a Leica VT1000S vibratome (Giacalone et al., 2021 (link)). The agarose was removed, and sections were then stained first with a rat anti-CD68 antibody (Bio-Rad), followed by DAPI (for visualization of nuclei) and an Alexa Fluor 647 goat anti-rat antibody. Stained samples were mounted with VECTASHIELD antifade mounting medium (Vector Laboratories). Images were acquired using a Leica SP8 confocal laser scanning microscope, with 0.5 μm z-steps and Leica LAS X software. 3D reconstruction was carried out with Volocity software as previously described (Sukumar et al., 2014 (link); Tan et al., 2013 (link)).