Feulgen-deoxyribonucleic acid analysis of rabbit aortic smooth muscle cells using scanning- integrating microdensitometry.
A procedure entailing the use of the Feulgen reaction is described for precise quantification of nuclear DNA levels in smooth muscle cells (SMC) of paraffin-processed microtome sections of the rabbit aorta. It was established that maximal, stable, and reproducible Feulgen-DNA (F-DNA) staining of SMC nuclei is achieved using 3.5 N HCl hydrolysis of 30-50 min prior to staining of aortic sections in Schiff reagent for 60 min at 22 degrees C. Scanning-integrating microdensitometry of Feulgen-stained SMC revealed that the tunica media is comprised of a relatively homogeneous population of cells with between 0.3 and 1% of the SMC nuclei yielding 3C or 4C (tetraploid) F-DNA levels, depending on location within the aortic wall. The nuclear chromatin in inner medial SMC was found to be in a more dispersed state than that of outer SMC (using nuclear area and nuclear susceptibility to acid hydrolysis as indices of chromatin dispersion). A linear correspondence was evidenced between nuclear area and nuclear F-DNA stainability throughout the tunica media. The observation that the lumenal portion of the tunica media contains a greater abundance of SMC with large, vesicular nuclei is interpreted as reflecting a greater metabolic reactivity of this compartment relative to that of SMC bordering the tunica adventitia.
D000818:Animals; D001013:Aorta, Thoracic / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis; D002843:Chromatin / Q000032:analysis; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D003720:Densitometry; D007962:Leukocytes / Q000032:analysis; D008099:Liver / Q000032:analysis; D008297:Male; D008853:Microscopy; D008938:Mitosis; D009131:Muscle, Smooth, Vascular / Q000032:analysis*; D011817:Rabbits; D012394:Rosaniline Dyes
D002843:Chromatin; D004396:Coloring Agents; C007722:Feulgen stain; D012394:Rosaniline Dyes; D004247:DNA