Tyramide Signal Amplification for Immunoelectron Microscopy

Abstract

Tyramide signal amplification (or catalyzed-reporter deposition) is an analyte-dependent reporter enzyme method that enhances the detection sensitivity of any given antigen by catalyzing the deposit of labeled tyramide that is covalently bound near the target molecule, providing high-density signal. Current methods allow identification of any protein or RNA using antibodies or probes, respectively. This technique has also been validated for electron microscopy detection by either DAB or for gold-conjugated streptavidin staining. Both techniques present several limitations, DAB staining hampers the ultrastructure of the sample since it precipitates all over the cell. Streptavidin, on the other hand, requires a strong permeabilization protocol which also compromises sample integrity. Hereby, we present a methodology which uses TSA in combination with immunogold labeling to improve the detection sensitivity for antigens that cannot be detected by conventional immunogold methods, or whose label is weak compared to background noise. As a proof of concept, we show the effectiveness of this technique for detecting difficult antigens expressed in the central nervous system such as NG2, PDGFRa, or BCAS1.