High‐pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43

Abstract

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.

Phosphorylation of Extrajunctional Cx43 in Ischemic-Preconditioned Rat Hearts

“…In other investigations, the hearts were analyzed under in vitro conditions [10,15,17] and were fixed with glutaraldehyde or formaldehyde prior to sectioning. These fixatives have been found to greatly reduce immunogenicity of connexins [19,20]. This fact may have contributed to the failure to detect any Cx43-lateralization, thus supporting the significance of methodical differences with respect to the present investigation.…”

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

“…During development, polarization of mechanical junctions at end-to-end contacts was observed before that of Cx43, which is initially widely distributed throughout the sarcolemma, including the lateral sides of the cell (Muhlfeld & Richter, 2006; Maass et al, 2007; Fromaget et al, 1992; Gourdie et al, 1991, 1992; Peters et al, 1994). Preferential Cx43 localization to IDs over developmental time was proposed to be a consequence of slower Cx43 internalization rates at IDs in comparison with other membrane areas, which are sparsely decorated with adhesion junctions and are thus less stable (Hirschy et al, 2006; Angst et al, 1997; Palatinus et al, 2012).…”

Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy

“…Some reports describe properties of immunocytochemical labeling of high-pressure frozen material (Chang et al 2003;Eppenberger-Eberhardt et al 1997;Kirschning et al 1998;McDonald 2007;McDonald and Müller-Reichert 2002;Monaghan and Robertson 1990;Monaghan et al 1998;Young et al 1995); however, only a few of them deal with quantitative data (Agarwal et al 2009;Bittermann et al 1992;Mistríková and Bednár 2010;Mühlfeld andRichter 2006, Müller-Reichert et al 2003;Sawaguchi et al 2004;Strádalová et al 2008). While several approaches and machines have been devised to cryofix satisfactorily the samples (Hawes et al 2007;Hess 2003;Hess et al 2000;Hohenberg et al 1994Hohenberg et al , 1996Jiménez et al 2006;Kiss et al 1990;Lancelle and Hepler 1989;Marsh et al 2001;McDonald 1999;Mims et al 2003;Müller and Moor 1984;Müller-Reichert et al 2008;Neuhaus et al 1998;Reipert et al 2004;Studer et al 1989;Wild et al 2005), the definition of FS protocols which would be optimal for immunolabeling has not been critically evaluated.…”