Axio scan z1 slide scanning fluorescence microscope

Mouse lungs were fixed with formaldehyde, paraffin embedded, and stained by multiplexed-ISH using optimized protocols, as previously described (39 (link), 40 (link)). Briefly, mouse lungs were directly placed into 4% PFA prepared in 1× PBS upon excision, fixed for 48 h at 4°C, and paraffin embedded. Next, 2-μm sections were cut from formalin-fixed paraffin-embedded (FFPE) blocks and mounted onto Superfrost Plus microscope slides (Fisher Scientific). ISH for M. tuberculosis 23S and pre-rRNA was carried out by RNAscope (Advanced Cell Diagnostics), which was performed as described earlier (39 (link), 40 (link)). Sequences for the M. tuberculosis-rRNA ISH probes were previously described (40 (link)). Whole-slide digital images were acquired at ×40 magnification using an Axio Scan Z1 slide scanning fluorescence microscope (Zeiss).

Mouse lung was formaldehyde-fixed and paraffin-embedded and stained by using multiplexed-ISH kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions^{47 (link)}. Specimens were directly placed into 4% PFA upon excision and fixed for 48 h at 4 °C before embedding. Next, 2 μm tissue sections were cut from FFPE blocks and mounted onto Superfrost Plus microscope slides (Fisher Scientific) prior to use. Multiplex-ISH was visualized after labeling with fluorescein isothiocyanate and cyanine 3.5 (^{47 (link)}). Whole-slide digital images were acquired at ×40 magnification using the Axio Scan.Z1 slide scanning fluorescence microscope (Zeiss) or at ×63 magnification using the SP8 laser scanning confocal system (Leica). Image analysis was performed using the ilastik machine-learning-based (bio)image analysis (www.ilastik.org)^{48 (link)}. The ilastik plugin for ImageJ (v. 2.1.0/1.53c) was used to export data from each region of interest in the ilastik HDF5 format.