Nanozoomer 2.0 ht whole slide imaging system

Lungs were harvested and fixed in 10% neutral buffered formalin (Fisher) for 3–4 days. Fixed lungs were then washed with PBS and dehydrated with serial incubation in 30%, 50%, and then 70% ethanol. Lungs were embedded, sectioned, and stained at the Pulmonary Morphology Core at Washington University. Dehydrated lungs were embedded in paraffin and sectioned at 5 μm using an RM2255 Fully Automated Microtome (Leica). Sections were collected onto charged microscope slides from a water flotation bath at 40°C. Slides were allowed to dry before staining. Sections were de-paraffinized with xylene; serially rehydrated in 100% alcohol, 95% alcohol, and then 70% alcohol; and washed briefly with distilled water. For H&E staining, sections were stained in Mayer hematoxylin solution, washed in warm running tap water, rinsed in distilled water, rinsed in 95% alcohol, and counter-stained in eosin Y solution. After staining, sections were dehydrated through 95% alcohol and then 100% alcohol, cleared through xylene, and mounted with Cytoseal 60 (Epredia). For AFB staining, sections were stained with Carbol Fuchsin and Fast Green Stain using an Acid Fast Bacillus Stain Kit (Epredia). Slides were imaged at the Alafi Neuroimaging Laboratory at Washington University using a NanoZoomer 2.0-HT Whole-Slide Imaging System (Hamamatsu) with a 40× objective.