Quantitative Lung Histopathology in Mycobacteria

Lungs were harvested from mice infected with Mabc for 10 days. Lungs were fixed in 10% formalin and embedded in paraffin wax. For histopathology, lung paraffin sections (4 µm) were cut and stained for hematoxylin and eosin (H&E) as previously described [21 (link)]. For analysis of the extent of tissue necrosis, propidium iodide (PI) staining was performed. For analysis of the extent of tissue necrosis, lung paraffin sections (4 µm) were cut and immunostained with propidium iodide solution (P3566; Invitrogen, Carlsbad, CA, USA). After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (LSM 710; Zeiss, CLSM, Jena, Germany), with constant excitation, emission, pinhole, and exposure-time parameters. H&E staining was scanned using an Aperio digital pathology slide scanner (Leica) and imaged using an Aperio ScanScope® CS System. To quantify the inflamed area and necrosis, the MFI of the red threshold was determined using FIJI software.