338 including thin-section TEM (Nagy et al. 1999, 2001; Ochalski et al. 1997; Yamamoto et al. 1990a, b), FRIL and, more recently, double-replica FRIL (DR-FRIL). [FRIL is the abbreviation originally introduced by Gruijters et al. (1987) for ‘‘fracture-replica-immunogold labeling’’ by a method not using the breakthrough SDS detergent washing procedure (Fujimoto 1995, 1997).] Alternative replica labeling procedures and nomenclature include SDS-FRL (Fujimoto 1995, 1997) and double-replica SDS-FRL (Li et al. 2008a). Our version of FRIL originally was distin- guished from SDS-FRL by the additional steps of Lexan plastic stabilization of replicas and by confocal grid map- ping to the limit of resolution of LM, prior to tissue removal by SDS washing and subsequent immunogold labeling (Rash et al. 1995, 1996). We found the added steps of FRIL to be essential for the analysis of gap junctions in complex CNS tissues. Regardless, we used both DR-SDS- FRL and DR-FRIL methods in this report. False Negatives Arising from LacZ mRNA Detection Methods Data from LacZ reporter systems have been interpreted as showing that a small percentage of neurons in the inner plexiform layer of the retina (i.e., cone bipolar cells) express only Cx45 and not Cx36 (Schubert et al. 2005), whereas the AII amacrine cells to which bipolar cells are known to couple reportedly express only Cx36. This appeared to pose a problem because Cx45 and Cx36 are reported to be ‘‘nonpermissive’’ for forming gap junctions (Teubner et al. 2000). Moreover, three groups using RT- PCR and immunocytochemistry (Han and Massey 2005; Lin et al. 2005) and immunoﬂuorescence of cryosections (Dedek et al. 2006) concluded that Cx45 and Cx36 were never coexpressed in the same neuron, with the further assertion (Han and Massey 2005) that Cx45 and Cx36 proteins are never detected in the same ﬂuorescent punc- tum. However, Dedek and coworkers reported that where Cx45 was present, it was present along with Cx36 in 30 % of puncta. Thus, those three groups separately concluded that because those cells are known to be coupled via gap junctions, their coupling required either (1) heterotypic coupling of Cx45 and Cx36 (i.e., that some additional factor allowed permissive coupling in vivo) or (2) that there must exist two additional connexin coupling partners to which Cx45 and Cx36 can separately couple. In our initial FRIL studies of Cx36 vs. Cx45 in 671 double-labeled gap junctions in the inner plexiform layer of rat and mouse retina (Li et al. 2008a), single-replica FRIL showed that ca. 90 % (607) contained only Cx36, whereas ca. 9 % (58) contained both Cx45 and Cx36 in the same hemiplaques (and a statistically insigniﬁcant \1 %, mostly very small, contained minimal labeling for Cx45 without 123 J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions labeling for Cx36). Those data demonstrated conclusively that the 58 neurons with hemiplaques containing labeling for Cx45 and Cx36 must have synthesized both Cx45 and Cx36 proteins, conceivable at different times (thereby possibly accounting for failure to detect one or the other connexin), or alternatively, suggesting that the failure to simultaneously detect both connexins in any neurons rep- resented a limitation of the detection method. Nevertheless, by single-replica FRIL, we were unable to ascertain whe- ther the coupling partners of the double-labeled gap junc- tion hemiplaques expressed either or both of those same connexins in the apposed hemiplaque. This question could be answered only by DR-FRIL, as developed and applied below. In this article we discuss the advantages of matched DR- FRIL in identifying the cellular types contributing speciﬁc connexins to neuronal gap junctions in retina and goldﬁsh hindbrain. Materials and Methods Animals used in this study were prepared under protocols approved by the Institutional Animal Care and Use Com- mittee of Colorado State University and conducted according to Principles of Laboratory Animal Care (U.S. National Institutes of Health publication 86-23, rev. 1985). These protocols included minimization of stress to animals and minimization of the number of animals used. One formaldehyde-ﬁxed rat retina and several formaldehyde- ﬁxed goldﬁsh hindbrains were prepared for DR-SDS-FRL and DR-FRIL, as previously described (Li et al. 2008a; Pereda et al. 2003). Fixed tissues were sliced to 150 lm thickness at 4 C in a refrigerated vibrating microslicer (DTK-1000; Dosaka, Kyoto, Japan) or a Lancer Vibratome 3000 (Leica Microsystems, Buffalo Grove, IL), inﬁltrated with 30 % glycerol as a cryoprotectant to reduce freezing damage by ice crystals, placed between two 4.6-mm gold planchettes and either frozen in a BalTec (BalTec, Balzers, Liechtenstein) 010 High-pressure Freezing Device (retina) or plunge-frozen in a mixture of 2:1 propane/ethane at -195 C (hindbrain). Sandwich samples were placed in a prototype double-replica device (Fig. 3a), the two planch- ettes were mechanically separated (fractured) and the two newly created mirror complements were coated with 2–5 nm of carbon and shadowed with *1.5 nm of plati- num, thereby creating matched double-replicas (Li et al. 2008a). These samples were either thawed, with the tissues picked up on copper ‘‘thin-bar grids’’ and mapped by reﬂectance microscopy (Fig. 3b, b0, showing matching complements of adult rat retina), or bonded to a gold ‘‘index grid’’ [not shown but see Pereda et al. (2003)] before SDS washing and immunogold labeling. Both