J. E. Rash et al.: Double-Replica FRIL Analysis of Gap Junctions 339 Fig. 3 Explanation of the DR-FRIL technique. a Photograph of the DR stage after fracturing of two specimens. Matched DR samples (indicated by B and B0 in a) are shown at higher magniﬁcation in b, b0), after ﬂoating off into buffer. b, b0 Replicated but undigested samples were mounted on thin-bar grids, with matching outlines indicated. Small tissue fragments were lost during washing (open outlines opposite corresponding outlined tissues). Bars occluding matching areas are indicated by dotted lines. c, c0 One of 11 pairs of matched gap junction hemiplaques (circles with inscribed quadrants) from the samples indicated in b, b0. Cx45 is labeled with 5-nm gold matched replica complements of retina were immungold- labeled simultaneously according to our previous descrip- tions (Li et al. 2008a), with 5-nm gold beads used to label Cx45 and 10-nm gold beads used to label Cx36. Matched replicas of goldﬁsh hindbrain were labeled with monoclo- nal MAB3045 (Biosciences Research Reagents; Millipore, Temecula, CA) against Cx35 and polyclonal rabbit anti- body against Cx34.7 [lot 2930-1 IL, from O’Brien et al. (2004)], with a single size of gold bead (5 nm) on goat anti-rabbit IgG for Cx45 and a different size of gold bead (10 nm) on goat anti-mouse IgG against Cx36. Results Procedures and Applications of DR-FRIL beads (lower right quadrants), whereas Cx36 is labeled with 10-nm gold beads (upper left three quadrants c, c0). Labeling for Cx45 is aligned with (i.e., opposite) labeling for Cx45 in the matching areas of the two hemiplaques. Likewise, labeling for Cx36 is aligned with labeling for Cx36. These matching hemiplaques demonstrate biho- motypic gap junctions. b, c Modiﬁed from Li et al. (2008a). d Diagram showing production of matched DRs and the subsequent immunogold labeling of bihomotypic plaques containing mostly Cx36 (white connexons labeled with large gold beads) and fewer Cx45 (black connexons, labeled with small gold beads) respects. First, both sides of the fracture plane are retrieved and, not incidentally, may be matched by laborious repeated examination and matching of corresponding ‘‘ﬁduciary marks’’ that nevertheless may be difﬁcult to recognize because they have opposite structural contour, or they may be covered by grid bars or damaged during SDS washing and labeling. Nevertheless, the rewards in obtaining deﬁnitive information regarding the connexin content, for example, of matched mirror complements are unequaled by any other technique. Equally important, a wide variety of scaffolding and accessory proteins can now be mapped and correlated with the biochemical composition and functional state of individual gap junctions in two apposed cells, as shown next. DR-FRIL Reveals Connexin Coupling Partners in Retinal Gap Junctions We have previously described the method of FRIL and details of its applications (Rash et al. 2001; Rash and Ya- sumura 1999). DR-FRIL differs from FRIL in several To determine whether Cx45-containing gap junctions are heterotypic vs. homotypic (and, hence, to identify connexin coupling partners in individual gap junctions), three cross 123