Figure 4.

Confocal fluorescence microscopy of lungs of mice transplanted with GFP-positive marrow cells and lungs of GFP-transgenic mice. (A) Hyperoxia-exposed recipient, Post-TPX Week 8 (P66). Several large-sized and irregularly-shaped GFP-positive cells are seen, contrasting with the smaller and more uniform SP-positive type II cells. Merging of the images shows co-localization of finely granular SP-positive material in the lower GFP-positive cell (arrow). SP-positive material in native type II cells is coarsely granular, consistent with intact lamellar bodies. Colocalization persisted at all three-dimensional volume angles analyzed. Volume slices along the xz (*) and yz (**) axes both show colocalization of green and red signals in the same cell, resulting in a yellow-orange composite signal and confirming the unequivocal presence of SP-positive material in this GFP-positive donor cell. (B) Hyperoxia-exposed recipient, Post-TPX Week 8 (P66). Apparent colocalization of an SP-positive granular structure is seen in a single GFP-positive cell (arrow). Three-dimensional volume analysis at various angles and x/y/z analysis of selected slices demonstrated that the SP-immunoreactive material is located outside of the GFP-positive cell, likely in an underlying type II cell (asterisks). This is an example of “overlay” artifact. (C) Age-matched GFP-transgenic mouse. Coarse granaular SP-positive structures, consistent with surfactant-containing lamellar bodies, are seen in GFP-positive (example shown by arrow) and GFP-negative (example shown by arrowhead) type II cells (pro–SP-C [Cy3, red] and GFP [fluorescein isothiocyanate, green] double immunofluorescence; original magnification: ×400, confocal fluorescence microscopy).