Figure 5.

Thin sections of OTC embedded, acetone-fixed lung immunostained with primary antibody to smooth muscle actin (5a), alpha keratin (5b) or CD31 (5c) and counterstained with DAPI. (a) Immunostaining is identified as dark pink in a pan-cytoplasmic distribution within smooth muscle cells around airways and blood vessels (arrows), consistent with high expression of this cytoskeletal protein in these cells. (b) Immunofluorescence for cytoskeleton component cytokeratin 7 is identified primarily in junctions between epithelial cells. (c) CD31 green fluorescence in the distribution of plasma membranes of endothelial cells (arrows identify small blood vessel and capillary endothelial cells). In each section, DAPI identifies the nuclei of cells, many of which are negative for the immunostain. The white arrows pointing to green PECAM-1 positive staining. The yellow arrow points to non-immunostained DAPI positive cells. (d) Because our confocal studies with immunostaining were performed on sections fixed with paraformaldehyde, and our images in figure1a-c were obtained from acetone fixed tissue, we performed an additional set of studies. 300 micron thick sections of tissue were fixed in paraformaldehyde, then immunostained with CD31 and counterstained with DAPI. Frozen sections of these tissues 4 microns thick were imaged with a Nikon Eclipse TE2000-U with a camera attachment (Qimaging QCAM FAST394) for epifluorescence. In these images, a pattern of plasma membrane uptake is evident in some (marked with white arrow) but not all (marker with yellow arrow) cells, similar to that in acetone fixed and OTC treated sections.