Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Ray et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.1031851100

Supporting Methods
Transmission Electron Microscopy.
Mouse lungs were perfusion-fixed through the heart with 2.5% glutaraldehyde in PBS. Lungs were removed and immersed in the same fixative overnight at 4ºC. Several 1 mm³cubes were removed from the lungs, washed in 3´ PBS, and then post-fixed in aqueous 1% OsO₄/1% K₃Fe(CN)₆ for 1 h. After 3 PBS washes, the pellet was dehydrated through a graded series of 30–100% ethanol/100% propylene oxide then infiltrated in 1:1 mixture of propylene oxide:Polybed 812 epoxy resin (Polysciences, Warrington, PA) for 1 h. After several changes of 100% resin over 24 h, the pellet was embedded in molds and cured at 37ºC overnight, followed by additional hardening at 65ºC for 2 more days. Ultrathin (60-nm) sections were collected on 200 mesh copper grids, stained with 2% uranyl acetate in 50% methanol for 10 min, followed by 1% lead citrate for 7 min. Sections were photographed using a JEOL JEM 1210 transmission electron microscope (Peabody, MA) at 80 or 60 kV onto electron microscope film (ESTAR thick base; Kodak, Rochester, NY) and printed onto photographic paper. Electron micrographs were digitized on a flatbed scanner at 400 ppi (StudioStar, Agfa). Digitized images were assembled into montages using Adobe PHOTOSHOP 6.0.