Tyramide signal amplification enhances the detectable distribution of connexin-43 positive gap junctions across the ventricular wall of the rabbit heart

Craig Steven McLachlan, Patricia R. Jusuf, Nicole Rummery, Sarah K. Kummerfeld, Brett Hambly, Mark A. McGuire, Virginia Turner

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)359-365
Number of pages7
JournalArchives of Histology and Cytology
Volume66
Issue number4
DOIs
Publication statusPublished - 1 Oct 2003
Externally publishedYes

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