Precision-cut mouse lung slices and immunostaining

We followed a previous publication (45) to prepare precision-cut mouse lung slices (PCLSs). Mice were euthanized with CO₂, and the chest cavity was opened to intratracheally inflate lung lobes with 2.5% low–melting point agarose. Mice were then kept at 4°C to solidify agarose. An inflated right upper lobe was excised from the mice and sliced with 150-μm thickness with Compresstome (VF-200-0Z, Precisionary) in ice-cold PBS. PCLSs were stained with an antibody cocktail for 1 hour at 4°C with gentle rocking, and without fixation, microscopic images were immediately acquired with a confocal microscope (Carl Zeiss 780 upright) using a 20× objective. Images were constructed with tile scans and z-stacks of images. The antibody panel for AM detection was used as follows: CD11c-PE, CD206-AF647, and CD326/EpCam-eFluor450.