Aque. Two gap junctions on unidentified membrane fragments (probable astrocyte fingers) that had partially overlapping clumps of gold beads for Cx have been designated as “false constructive labeling” or “noise” (Rash and Yasumura H,H) and,therefore,have been excluded from this analysis. All major antibodies had been diluted to gml. Immediately after main labeling,replicas had been rinsed and counterlabeled for h employing many combinations of goat antirabbit immunoglobulinG (IgG) conjugated to nm andor nm gold beads (Jackson ImmunoResearch Laboratories,Inc West Grove,PA,USA) and goat antimouse IgG,conjugated to nm gold beads (Jackson ImmunoResearch),as Mikamycin B outlined by our detailed methods (Kamasawa et al. Samples doublelabeled for NR and panAMPA have been labeled with and nm gold,respectively (both from Chemicon International Inc as reported in Rash et al. Chemicon antibodies now offered from EMD Millipore).Table Twenty numbered antiCximmunogoldlabeled and gridmapped gap junctions in adult rat hippocampus,with their anatomical places,size ranges (number of connexons,if countable),and their associations with labeled and unlabeled glutamate receptor PSDs (as tight patches of nm Eface IMPs) vs. dispersed nm Eface IMPs that happen to be believed to represent extrasynaptic or “reserve” receptors. GJ location No. of GJs GJ size variety GJs close to Eface PSDs Dentate gyrus Hilus CA lucidum CA oriens CA oriens CA radiatumH H H GJs near clusters of nm Eface IMPs H H,H H H,H H H,H H,H,H H H,H H H H,H,H H H,H H H GJs on spines GJs on MF axon terminals Dendrodendritic GJs GJs in unidentified cell pairsAlso indicated are gap junctions where distinct distinguishing features had been not identifiable in either neuron. Some numbered gap junctions seem in multiple columns. GJ,gap junction; size range,quantity of connexons,from smallest to largest; PSD,postsynaptic density; IMP intramembrane particle; SVs,synaptic vesicles; with ,the other cell not identified.Frontiers in Neuroanatomywww.frontiersin.orgMay Volume Post HamzeiSichani et al.Glutamatergic mixed synapses in hippocampusAfter labeling and rinsing but ahead of TEM viewing,the immunogoldlabeled samples had been coated a third time with nm of carbon around the labeled side to: (a) surround and immobilize gold beads,(b) anneal thermalexpansion cracks in the replica before removal of the Lexan help film,thereby helping to retain replica integrity,and (c) stop displacement of the replicas with respect to the grid throughout subsequent removal from the Lexan assistance film. The Lexan help film was then removed by immersing the grids in dichloroethane for h. FRIL samples were examined at kV in a JEOL EXII TEM (JEOL,USA). Stereoscopic images obtained with an integrated angle have been made use of for: (a) assessing complex D membrane topography,(b) confirming that every immunogold bead was on the tissueside from the replica (Rash and Yasumura,,and (c) discriminating the smaller sized (nm) gold beads in the similarly electronopaque platinum caps on to nm IMPs (Pereda et al. Rash et al. Each and every immunogoldlabeled gap junction found by FRIL was examined at multiple tilts,and where needed,at multiple rotations (i.e to receive optimum views of vital features,including PSDs PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24893121 and interiors of spines). Freezefractured neuronal and glial processes have been identified as outlined by our published criteria (Rash et al . FRIL TEM negatives were digitized by an ArtixScan f digital scanner (Microtek; Santa Fe Springs,CA,USA) and processed using Adobe Photoshop CS (Adobe Systems,San Jose,CA,USA),.