Figure 8. Optical Clearing and 3D Volumetric Analyses Delineate the Architecture of the Entire CCS and SA Nodal Substructure Within Intact Murine Hearts.

(A) Schematic representation of workflow for iDISCO+ clearing of intact embryonic and postnatal mouse hearts and visualization using light sheet microscopy. At least 10 biological replicates were used for each immunolabelling. (B) A wild-type whole heart from an E16.5 mouse embryo is shown at four angles of view (0, 90, 180 and 270 degrees) at 0.63x magnification. Top and bottom rows are the same optically cleared heart using iDISCO+ where, in the top row, background fluorescence has been saturated to provide a representation of the opacified heart. Bottom row demonstrates the same tissue-cleared heart, showing Igfbp5 immunostaining which marks the cardiac conduction system in its entirety. (C) iDISCO+ cleared wild-type, intact SAN (6.3x magnification) from E16.5 murine heart co-immunolabeled for Hcn4 (red) and Rgs6 (green) protein. SAN shown at three angles of view (0, 90, ~225 degrees). (D) Merge image (Hcn4 in red, Rgs6 in green) with two major transitional sinoatrial conduction pathways (SACPs) outlined (Hcn4^neg/Rgs6⁺). Purple SACP: from SAN body directed rightward; Black SACP: from the SAN head directed inferiorly and leftward. A third Hcn4+ SACP is also marked by Rgs6 and emerges inferiorly from the tail of the SAN to give rise to the internodal tracks (blue line) not seen here but visible in panel 8B. Ant, Anterior. AVN, atrioventricular node. His, His bundle. INT, internodal tracks. LA/RA, left or right atrium. LAVRB, left AV ring bundle. LBB/RBB, left of right bundle branch. LV/RV, left or right ventricle. PF, Purkinje fiber. Post, Posterior. RAVRB, right AV ring bundle. SAN, sinoatrial node.