title: Tyramide Signal Amplification for Immunofluorescent Enhancement.
abstract: Enzyme-linked signal amplification is a key technique used to enhance the immunohistochemical detection of protein, mRNA, and other molecular species. Tyramide signal amplification (TSA) is based on a catalytic reporter deposit in close vicinity to the epitope of interest. The advantages of this technique are its simplicity, enhanced sensitivity, high specificity, and compatibility with modern multi-label fluorescent microscopy. Here, we describe the use of a TSA kit to increase the signal of enhanced green fluorescent protein (eGFP) expressed under the control of Slc17a6 regulatory elements in the brain of a transgenic mouse. The labeling procedure consists of 6 basic steps: (1) tissue preparation, (2) blocking of nonspecific epitopes, (3) binding with primary antibody, (4) binding with horseradish peroxidase-conjugated secondary antibody, (5) reacting with fluorescent tyramide substrate, and (6) imaging of the signal. The procedures described herein detail these steps and provide additional guidance and background to assist novice users.
authors.affiliation: Department of Neurosciences, School of Medicine, University of California San Diego (UCSD), 9500 Gilman Drive, La Jolla, CA, 92093, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Faget
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Hnasko
mesh_terms: D000577:Amides; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001921:Brain / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect / Q000295:instrumentation / Q000379:methods*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry; D018796:Immunoconjugates / Q000737:chemistry*; D051379:Mice; D008822:Mice, Transgenic; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014439:Tyramine / Q000737:chemistry*; D050599:Vesicular Glutamate Transport Protein 2 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; D000906:Antibodies; D000939:Epitopes; D018796:Immunoconjugates; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins; C493477:Slc17a6 protein, mouse; D050599:Vesicular Glutamate Transport Protein 2; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006735:Horseradish Peroxidase; D014439:Tyramine