Immunofluorescence for cytokeratins and direct fluoresence analysis for eGFP identifies donor-derived transgene-expressing epithelial cells in the lung. (a­d) Lung tissue 2 mo after transplantation of lethally-irradiated recipients with transduced bone marrow cells shows eGFP⁺ cells. eGFP fluoresces in the FITC (green) channel but not in the Cy3 channel (not shown), nuclei are stained with DAPI (blue), and cytokeratins are immunostained with alexa fluor 568 (red). (e­h) The same images are shown in a-d without the green channel. When fluoresence in the green channel is removed, it is clear that the cytokeratin staining is expressed in several eGFP-positive cells (arrows). eGFP can diffuse to the nucleus and is therefore found throughout the cell. (a, e) Pneumocytes form rings of alveolar tissue. Two cells are eGFP-positive. The bottom center cell is cytokeratin-positive and the top left cell appears to be, although is not definitely, cytokeratin-positive (arrows). (b, f ) Two eGFP-positive, cytokeratin-positive, bone marrow­derived lung cells are evident. (c, g) Enlarged image of an eGFP-positive, cytokeratin-positive cell. (d, h) Two adjacent eGFP-positive cells are also positive for cytokeratin (arrow), whereas others are clearly cytokeratin-negative (arrowheads). Original magnification: ×60. For more information see article by Grove and coworkers on page 645.