Technical considerations in evaluating the endothelial integrity of rat aortic preparations with silver staining

Assessment of endothelial integrity is an obligatory step in many pharmacological studies. Integrity of endothelium is affected by manipulations performed during the removal and cleaning of the vessel and by some of the silver-staining techniques utilized for demonstrating interendothelial junctions. When aortas were cleaned of periadventitial tissue in cold Tris-saline (once separated from the animal) by untrained personnel, only 45% of the endothelium was preserved. When cleaning was performed in situ by trained personnel while flushing with cold Krebs-Ringer-6% albumin, over 95% was left intact. AgNO3-staining performed before fixation produced a 50% loss of endothelium when using NH4Br and (NH4)2S as developers. AgNO3-staining performed after fixation produced over 95% recuperation of endothelium when 2% glutaraldehyde, 150 mM NaCl, 40 mM phosphate buffer, pH 7.4, were utilized as initial fixative, NH4Br and (NH4)2S being equally effective as developers. Chloride ions were necessary to intensify silver lines. Several patterns of deendothelization were produced by mechanical and chemical injury with saponin, NH4Br and (NH4)2S. In all cases, hematoxylin staining was employed as an auxiliary technique to interpret images of injured endothelium. Presence of albumin protected the endothelium from mechanical damage.