Signal amplification at the ultrastructural level using biotinylated tyramides and immunogold detection.
The tyramide amplification technique has recently been developed for signal enhancement in enzyme-linked immunosorbent assays and western blots. This method relies on using labelled tyramides as substrates for peroxidase, resulting in an immobilization of the labelled tyramide residues (tyramide reaction). We succeeded in establishing reliable protocols for the use of the tyramide reaction at the electron microscopic (EM) level. As model systems we chose the visualization of DNA in late spermatocytes, of actin in skeletal muscle, and the visualization of an rDNA probe after DNA-DNA in situ hybridization. We observed a significant increase in signal density after performing the tyramide reaction at the EM level. The tyramide amplification technique at the ultrastructural level therefore appears to be a useful tool to detect even a few epitopes present at the surface of a section as shown after in situ hybridization. It offers advantages over other amplification systems, such as the peroxidase-mediated deposition of diaminobenzidine, because of an increased spatial resolution, whereas specificity and sensitivity are comparable to the conventional immunogold detection method.
Schöfer
C
C
Institute for Histology and Embryology of the University of Vienna, Austria. christian.schoefer@univie.ac.at
Weipoltshammer
K
K
Almeder
M
M
Wachtler
F
F
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D002417:Cattle; D004247:DNA / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008853:Microscopy; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000378:metabolism; D012680:Sensitivity and Specificity; D013087:Spermatids / Q000737:chemistry; D014439:Tyramine / Q000737:chemistry*
D000199:Actins; D004247:DNA; D014439:Tyramine