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Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain
Author(s) -
Nagy J. I.,
Yamamoto T.,
Sawchuk M. A.,
Nance D. M.,
Hertzberg E. L.
Publication year - 1992
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.440050102
Subject(s) - immunoperoxidase , immunostaining , biology , blot , immunohistochemistry , central nervous system , endocrinology , biochemistry , antibody , immunology , monoclonal antibody , gene
We have shown by immunohistochemical methods that the gap junction protein connexin43 is heterogeneously distributed in rat brain (Yamamoto et al: J Comp Neurol 302:853, 1990). Here we have compared quantitatively the relative amount of connexin43 detected on Western blots of seven central nervous system (CNS) regions with the density of connexin43‐immunoperoxidase reactivity in these regions. As has been observed on Western blots of several cell types, homogenates of these CNS regions contained two forms of connexin43, its dephospho form with an apparent mobility of ∼41 kDa and its ∼3 kDa phosphorylated form. While the relative quantities of connexin43 varied considerably among the brain regions, the ratio of the 43/41 kDa forms, 0.71, was relatively uniform (correlation coefficient, r = 0.92). Sections of brain processed for connexin43‐immunolocalization by the peroxidase–antiperoxidase (PAP) method showed that chromogen deposition was linear with incubation time in reaction medium. Optical density of tissue connexin43‐immunoreactivity in each of the seven areas plotted against the density of connexin43 bands on Western blots gave a correlation coefficient of r = 0.90. Connexin43‐immunoreactivity had a similar appearance in sections processed by PAP or immunofluorescense procedures and consisted of isolated or aggregates of puncta. For the brain regions examined we conclude: (1) that although total levels of connexin43 vary widely, the levels of its phosphorylated and non‐phosphorylated forms are held in fairly constant proportion; (2) that there is a high correlation between the relative amounts of connexin43 detected biochemically and the density of connexin43‐immunostaining in tissue sections; and (3) that immunohistochemical staining for connexin43 reflects the relative amounts of connexin43 and possibly the numbers of gap junctions present in individual brain areas.