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Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve (2002)

Li, Xinbo ; Lynn, B. D. ; Olson, C. ; [et al.]

Oxford, UK : Blackwell Science, Ltd

European journal of neuroscience 16 (2002), S. 0

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ISSN: 1460-9568

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Medicine

Notes: The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) ‘rosettes’ and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1460-9568.2002.02149.x

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Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis (1999)

Rash, J. E. ; Yasumura, Thomas

Springer

Cell & tissue research 296 (1999), S. 307-321

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ISSN: 1432-0878

Keywords: Key words Freeze fracture ; Immunogold labeling ; Connexins ; Aquaporin-4 ; Brain ; Spinal cord

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a ”control” tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.