Techniques are described for visualizing intracellular tropoelastin at the light level using immunofluorescence and immunogold techniques. Best results were obtained with B5 fixative on cells permeabilized with acetone. Using either formaldehyde or paraformaldehyde for fixation (instead of B5) resulted in both less reproducible and less intense intracellular staining, and permeabilization of the cells with ethanol resulted in relatively high background staining compared with that obtained with cold (-20 degrees C) acetone. Intracellular tropoelastin was seen most prominently in the perinuclear region, and the intensity of staining agreed with the reported rate of tropoelastin synthesis as assayed by enzyme-linked immunosorbent assay (ELISA) and RNA hybridization studies. The applicability of the intracellular staining technique for studying the elastin phenotype was tested by demonstrating increases in both the number of positive cells and in the intensity of elastin staining in cells treated with smooth muscle elastogenic factor (SMEF), an elastogenic factor known to stimulate elastin production.
|Number of pages||5|
|Journal||American Journal of Respiratory Cell and Molecular Biology|
|State||Published - Jul 1990|