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Proof of myocardial autophagy by combining antigen retrieval and the avidin–biotin peroxidase complex method

      The cardiomyocytes of patients with dilated cardiomyopathy (DCM) show autophagic changes [
      • Vigliano C.A.
      • Cabeza Meckert P.M.
      • Diez M.
      • et al.
      Cardiomyocyte hypertrophy, oncosis, and autophagic vacuolization predict mortality in idiopathic dilated cardiomyopathy with advanced heart failure.
      ,
      • Shimomura H.
      • Terasaki F.
      • Hayashi T.
      • Kitaura Y.
      • Isomura T.
      • Suma H.
      Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy.
      ]. Membrane-bound vacuoles containing degenerated cytoplasmic organelles [
      • Shimomura H.
      • Terasaki F.
      • Hayashi T.
      • Kitaura Y.
      • Isomura T.
      • Suma H.
      Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy.
      ] have been observed by electron microscopy in the cardiomyocytes of DCM patients using immunogold staining for ubiquitin, suggesting an autophagic process [
      • Kostin S.
      • Pool L.
      • Elsässer A.
      • et al.
      Myocytes die by multiple mechanisms in failing human hearts.
      ]. In monitoring autophagy, it is necessary to confirm the expression of microtubule-associated protein light chain 3 (LC3), which is a marker of autophagosome binding to membranous structures associated with autophagy such as phagophores and autophagosomes [
      • Kabeya Y.
      • Mizushima N.
      • Ueno T.
      • et al.
      LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
      ]. However, previous pathological studies using an indirect immunohistochemical technique [
      • Vigliano C.A.
      • Cabeza Meckert P.M.
      • Diez M.
      • et al.
      Cardiomyocyte hypertrophy, oncosis, and autophagic vacuolization predict mortality in idiopathic dilated cardiomyopathy with advanced heart failure.
      ,
      • Kostin S.
      • Pool L.
      • Elsässer A.
      • et al.
      Myocytes die by multiple mechanisms in failing human hearts.
      ] deemed such vacuoles as autophagosomes only. In addition, images taken by immunogold electron microscopy are of low quality, and the technique relies on fresh samples of cardiac tissue being available. Moreover, formalin fixation and paraffin embedding often destroy most or all of the native LC3 immunopositivity in heart tissue samples.

      Keywords

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