title: Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining.
abstract: Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin-biotin-peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining.
authors.affiliation: Institute of Anatomy I, University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Horling
authors.affiliation: Institute of Anatomy I, University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: W L
authors.initials: WL
authors.lastname: Neuhuber
authors.affiliation: Institute of Anatomy I, University of Erlangen-Nuremberg, Erlangen, Germany. Electronic address: marion.raab@anatomie1.med.uni-erlangen.de.
authors.forename: M
authors.initials: M
authors.lastname: Raab
mesh_terms: D000818:Animals; D016477:Artifacts; D001665:Binding Sites; D004947:Esophagus / Q000378:metabolism*; D005189:False Positive Reactions; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D019809:Streptavidin / Q000378:metabolism*; D014018:Tissue Distribution; D014439:Tyramine / Q000378:metabolism*
chemical_list: D019809:Streptavidin; D014439:Tyramine