Precision-cut lung slices were made from neonatal mouse lungs at P5 or P12 or from adult mouse lungs using previously published methods (62, 63). Briefly, after euthanasia, lungs were perfused with cold PBS, after which a blunt-tipped catheter was inserted into the trachea. Low melting temperature agarose (2% agarose in DMEM/F12 1:1) was then instilled into the lung via the tracheal catheter, after which the lung was removed en bloc and placed on ice. Using a Leica VT1000E vibrating microtome tissue slicer, 250 μm thick sections were cut from the left lobe. Tissue slices were cultured in DMEM/F12 with or without HNE treatment for up to 96 hours at 37oC, after which lung slices were harvested and fixed in 10% formalin for additional studies.

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