Endothelin immunocytochemistry: indications of false-positive labeling patterns and non-detectable antigen concentrations

Abstract

Endothelin is an endothelium-derived peptide with potent vasoconstrictor and mitogenic properties. Since studies concerning the immunocytochemical localization of endothelin are often inconsistent we tried to clear up some of these discrepancies by comparing specificity and labeling patterns of different endothelin antibodies. Monoclonal and polyclonal endothelin antibodies ( n=7) were examined concerning their reactivity with endothelins and heart tissues by immunoblotting. Using immunofluorescence microscopy reactivities with human non-failing hearts, failing hearts, and cultured endothelial cells were examined. Specificity of labelings was assessed by absorption controls and functional controls using endothelial cells conditioned to produce different amounts of endothelin. As shown by immunoblotting five out of seven endothelin antibodies revealed both specific endothelin reactivity and negligible non-specific reactivities with heart proteins. Immunocytochemistry showed vascular reactivity of N-terminal endothelin antibodies to be associated with alpha-smooth muscle actin expression. One N-terminal antibody showed additional nuclear reactivity, and one C-terminal antibody vimentin-like labeling patterns. Although these reactivities were abolished in absorption controls these labelings had to be graded non-specific because functional specificity of endothelin antibodies could not be proven. The remaining antibodies revealed no endothelial reactivity even after tyramide signal amplification. Cellular endothelin concentrations ranged around 2,000-fold below the detection limit of immunocytochemical methods. Discrepancies in endothelin immunocytochemistry may originate from false-positive results and from expression levels of endothelin below the detection limit of immunocytochemical methods.

Controls for Immunocytochemistry

“…Examples of such false negatives occur when the epitopes are shared on several antigens and bind to all antigens by absorption (e.g., Swaab et al 1977). To ensure a negative absorption control is correct, an immunoblot must show that the epitope is found on a single protein (e.g., Wolf et al 2001). Thus, the absorption control needs an additional control to eliminate the possibility that the primary antibody is bound to multiple antigens.…”

Graft-infiltrating Dendritic Cells and Coronary Endothelial Dysfunction After Human Heart Transplantation

“…Fluorescence immunostaining has been described in detail elsewhere. 10 In brief, heart cryosections (5 m thick) mounted on glass slides were exposed to 4% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) consisting of 10 mmol/liter phosphate buffer and 150 mmol/liter NaCl for 5 minutes. Preparations were rinsed consecutively with distilled water, PBS containing 50 mmol/liter glycine, and PBS complemented with 0.5% bovine serum albumin and 0.2% gelatin (PBG).…”

Progress in focus: recent advances in Histochemistry and Cell Biology

“…Endothelins are endothelium-derived peptides with potent vasoconstrictor and mitogenic properties (Wolf et al 2001). Two distinct G-protein-coupled endothelin receptors have been cloned, ET A and ET B .…”