Fluorochrome-labeled Tyramides: Use in Immunocytochemistry and Fluorescence In Situ Hybridization

Abstract

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

“…Thus tyramine is readily conjugated with a variety of hapten labeled esters such as FITC-ester, and biotin-ester by simply mixing the same mole of each component in DMSO [5,6]. Biotin-tyramide and FITC-tyramide have been applied for post ISH amplification after several different ISH methods, including DNA/mRNA ISH and chromosomal ISH with a variety of probe types [9,20,26,30,40,46,47,56,[59][60][61]. In the case of using biotin-tyramide, biotinylated probe is sequentially reacted with primary HRP conjugated streptavidin, 30ug/ml of biotintyramide in the presence of 0.001% H2O2, and secondary HRP conjugated streptavidin (Fig.…”

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

“…The green fluorescent protein (GFP) was discovered as a by product of isolating aequorin from jelly fish by Shimomura et al [1] in 1962. The importance of the discovery was not obvious until much later; GFP proved to be an excellent protein marker molecule for gene expression (see [2]).…”

“…The field has been plagued by controversy mostly due to differences in techniques used by the different groups to follow cell fate. In the last decade a new, very sensitive immunocytochemical technique became available utilizing tyramide signal amplification [1]. Since we also noticed very faintly fluorescence green cells in our experimental samples we decided to try the most sensitive technique to visualize most of the GFP expressing cells.…”

“…The field has been plagued by controversy mostly due to differences in techniques used by the different groups to follow cell fate as summarized in [21]. In the last decade a new, very sensitive technique became available utilizing tyramide signal amplification [22,23] and its application to immunohistochemistry was reported [1] describing dilutions of primary antibodies for optimal immunohistochemistry [24] as well as its use in dual immunostaining techniques [25]. Since we also noticed very faint green fluorescent cells in our experimental samples we decided to apply this technique to attempt to visualize most of the GFP expressing cells.…”

Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection

“…There are, however, numerous examples where important biological markers for cancer, infectious disease, or biochemical processes are present at too low a concentration in body fluids or tissues to be detected by using conventional immunoassays. Recent advances in the field of low-level Ag detection include the development of stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescencebased staining and immunoblotting (2), and the application of signal amplification methods such as tyramide deposition (3). Although these techniques can be quite powerful, greater sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when Ag density is extremely low.…”