title: Tyramide signal amplification enhances the detectable distribution of connexin-43 positive gap junctions across the ventricular wall of the rabbit heart.
abstract: Previous mapping studies examinig the distribution and pattern of staining for connexin-43 expression (the major ventricular gap junction protein) across the ventricular wall have yielded variable findings. The aim of this study was to determine if variations in the distribution of connexin-43 were due to histochemical detection problems, i.e. cross-linking of antigenic sites as a consequence of aldehyde fixation and/or due to low levels of protein expression within the epicardial or endocardial regions of the heart. Immunoperoxidase staining of connexin-43 using the ABC method was carried out in crosssections of rabbit hearts at the level of the papillary muscle. The following treatments were examined: the antibody (Ab) only, Ab with 1/2 Tyramide Signal Amplification (TSA) or full TSA; antibody with microwave antigen retrieval (AR); Ab + 1/2 TSA + AR and finally Ab + TSA + AR. Under light microscopy and using computerized image analysis the percentages of ventricular cross-sectional transmural staining for the different treatment groups were calculated: Ab amounted to only 55%; Ab + 1/2 TSA 63%; Ab + TSA 78%; Ab + AR 72%; Ab + AR + 1/2 TSA 72% and Ab + AR + TSA 88%. The percentages of transumural connexin-43 staining in both TSA + Ab and Ab + TSA + AR groups when compared to Ab only were significantly greater p < 0.01. The antigenic cross-linking due to aldehyde fixation and low levels expression of connexin-43 are contributing factors that influence the immunohistochemical detection of connexin-43 in the mammalian heart. Methodological enhancement for the detection of connexin-43 in this study was derived primarily from amplification of low background levels of connexin-43 being expressed using the TSA protocol. This is supported by the significant differences encountered when TSA was utilized in the protocol and compared with antibody treatment only.
authors.affiliation: Department of Cardiology, University of Sydney, Sydney, Australia. reperfusion@hotmail.com
authors.forename: Craig S
authors.initials: CS
authors.lastname: McLachlan
authors.forename: Patricia R
authors.initials: PR
authors.lastname: Jusuf
authors.forename: Nicole
authors.initials: N
authors.lastname: Rummery
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Kummerfeld
authors.forename: Brett
authors.initials: B
authors.lastname: Hambly
authors.forename: Mark A
authors.initials: MA
authors.lastname: McGuire
authors.forename: Virginia
authors.initials: V
authors.lastname: Turner
mesh_terms: D000818:Animals; D000941:Antigens / Q000737:chemistry; D001011:Aorta / Q000473:pathology; D018031:Connexin 43 / Q000096:biosynthesis*; D017629:Gap Junctions; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D009206:Myocardium / Q000378:metabolism; D010210:Papillary Muscles / Q000378:metabolism; D011817:Rabbits; D014439:Tyramine / Q000494:pharmacology*
chemical_list: D000941:Antigens; D018031:Connexin 43; D014439:Tyramine