Quantitative Assessment of Lung Cell Distribution

All quantitative assessments of BRM cell or PC distribution, including calculations of clustering L values and cell densities in the alveoli or in uninfected and infected sites, were done by analyzing large tiles of static lung sections using two-photon excitation laser scanning microscopy (TPLSM), as previously described (MacLean et al., 2022 (link)). Briefly, after euthanization with pentobarbital, mouse lungs were inflated with 1 ml of 30–35°C low-melt agarose (1% in PBS) into the lungs. Individual lobes were dissected and stored in PBS before sectioning at 450 mm on a vibratome (Campden Instruments Ltd.). Sections were mounted on plastic coverslips using Vetbond tissue glue (3M), placed on ice in PBS, and immediately imaged. Sections were imaged on a Zeiss LSM880 microscope, using a primary excitation wavelength of 930 nm, with an Apochromat 20 × 1.0 DIC VIS-IR D = 0/0 (UV) objective lens.