J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions connexins on the oligodendrocyte side (i.e., Cx32, Cx47) (Altevogt et al. 2002; Altevogt and Paul 2004; Kleopa et al. 2004; Li et al. 1997; Nagy et al. 2004; Nagy and Rash 2000; Scherer et al. 1995). Recent studies of coupling permissiveness in N2A cells expressing glial connexins indicate that, in addition to permissive homotypic combi- nations (e.g., Cx30/Cx30, Cx43/Cx43, Cx32/Cx32 and Cx47/Cx47), functional coupling can occur between Cx30 and Cx32 and between Cx43 and Cx47 (Orthmann-Murphy et al. 2007), as well as between Cx30 and Cx47 (Magnotti et al. 2011). Finally, homologous cell types (two neurons, for example) expressing two different connexins at the same gap junction plaque may also form either bihomo- typic or heterotypic gap junctions. These several coupling conﬁgurations may provide vertebrate gap junctions with the molecular basis for attaining functional diversity, including electrical rectiﬁcation (directionality of current ﬂow), which has been proposed to require molecular asymmetry of apposed connexon hemichannels (Barrio et al. 1991; Palacios-Prado and Bukauskas 2009; Rubin et al. 1992; Verselis et al. 1994). Currently, these func- tionally distinct conﬁgurations of connexins within apposing hemiplaques can be distinguished in vivo only by the double-replica immunogold labeling technique, as described below. Sources of Ambiguity in Previous Approaches False-Positive Identiﬁcation of Protein Expression in Neural Cells False-positive identiﬁcations of connexin protein expres- sion in cells and misassignment of those connexins to an inappropriate cell type can occur for three main reasons. The ﬁrst is inadequate conﬁrmation of anticonnexin anti- body speciﬁcity, which can result in failure to recognize off-target labeling of proteins. The use of connexin knockout (KO) mice can be considered the ‘‘gold standard’’ for conﬁrmation of speciﬁcity. Many of the antibodies that we use have been characterized for speciﬁcity by com- parison of immunoﬂuorescence and/or immunoblotting results in wild-type mice vs. mice with KO of the various connexins. For connexins relevant here, such character- ization has included antibodies against Cx26 (Nagy et al. 2011), Cx30 (Lynn et al. 2011), Cx32 (Nagy et al. 2003), Cx36 (Li et al. 2004), Cx47 (Li et al. 2008b) and Cx57 (Ciolofan et al. 2007). In addition, where possible, we have included the use of two different antibodies generated against different sequences within individual connexins, as well as the use of both rabbit polyclonal and mouse monoclonal antibodies generated against the same sequence in some of the connexins. The speciﬁcity of 335 antibodies against Cx43 and Cx45 has been established ultrastructurally by showing their detection in gap junction plaques in identiﬁed cell types (Rash et al. 2001). A second source of error derives from the failure to detect a protein target even when using antibodies with proven speciﬁcity and proven immunohistochemical applications. This can arise mainly from nonoptimal tissue preparation for immunostaining, particularly if prescribed ﬁxation protocols are not followed. We have previously emphasized that immunoﬂuorescence detection of some connexins requires very weak ﬁxation conditions, where overﬁxation results in reduced detection or abolition of immunolabeling entirely (Li et al. 2008b). The third source of connexin misassignment arises from the limited resolution of light microscopy (LM). Because of inherent limits of LM resolution, current immunocyto- chemical methods applied to complex CNS tissues are unable to discern whether speciﬁc connexins, reportedly identiﬁed either by diffuse cytoplasmic staining (Colwell 2000) or by the presence of both punctate immunolabeling for connexins and widespread cell-surface immunoﬂuo- rescence (Nadarajah et al. 1996, 1997), link either neurons or glia or both. When only a single cell type (neuron) is visualized by immunoﬂuorescence in CNS tissue, without companion bright-ﬁeld or differential interference optics to reveal glial cells (Fig. 2a), it is not possible to assign connexins unambiguously to the visualized neuron, regardless of apparent close proximity of connexin labels. This failure to account for CNS tissue complexity is implicit in representative thin-section transmission electron microscopic (TEM) images (Fig. 2b), wherein all spaces between neurons are seen to be completely ﬁlled with the two primary types of macroglial cells (astrocytes and oli- godendrocytes) found throughout the neuropil and by their even more pervasive thin processes that are also below the limit of LM resolution. For further clariﬁcation, the limits of resolution in the blue and red wavelengths are super- imposed on the TEM image (Fig. 2b, blue and red discs), revealing that a single pixel at the limit of LM resolution in those wavelengths would overlap multiple plasma mem- branes of multiple cell types, with the blue dot overlapping with a neuronal plasma membrane, two astrocyte ﬁngers and an oligodendrocyte soma and nucleus. This image suggests that, in the absence of companion ultrastructural examination, complex interdigitations of neuronal and glial processes preclude or make questionable the LM assign- ment of speciﬁc connexins to speciﬁc cell types in con- voluted CNS tissue. Of course, this problem of assigning proteins to speciﬁc cell margins applies equally well to subcellular localization of all other membrane proteins. To investigate the basis for putative neuronal gap junc- tions reportedly containing Cx26, Cx32 and Cx43 by freeze- fracture replica immunogold labeling (FRIL) using knife- 123