After 5?moments of etching at room heat (different times were also tested) sections were rinsed with 96% ethanol three times, then with DW. sections with Na-ethanolate and treating them with SDS dramatically increase the labeling efficiency of synaptic proteins. We also demonstrate that this method is ideal for the molecular characterization of individual synapses following paired recordings, two-photon [Ca2+] or glutamate-sensor (iGluSnFR) imaging. This method fills a missing space in the toolbox of molecular and cellular neuroscience, helping us to reveal how molecular heterogeneity prospects to diversity in function. hybridization, single-cell RT-PCR, single-cell RNA sequencing [RNA-seq], and proteomic analysis of synaptosomes; Fuzik et?al., 2016; Gall and Pardue, 1969; Takamori et?al., 2006; Wang et?al., 2009). They are ideal for investigating the molecular mechanisms underlying differences in the functional properties of unique synapse populations (e.g., cerebellar parallel fiber versus calyx of Held synapses). In these synapses, the?pre- and postsynaptic elements belong to distinct cell classes with unique gene expressions. The up- or downregulation of genes in combination with functional analysis have been successfully used to reveal the functions of many synaptic proteins (examined by Sdhof, 2012). However, not only unique synapse populations possess different functional properties. It has been demonstrated that this subunit composition of GluRs on a single postsynaptic cell?can be presynaptic input dependent (Fritschy et?al., 1998; Rubio and Wenthold, 1997; Watanabe et?al., 1998). The functional properties and molecular composition of AZs of a single presynaptic cell could depend around the postsynaptic target cell type (Ali and Thomson, 1998; ltes et?al., 2017; Losonczy et?al., 2002; Pouille and Scanziani, 2004; Shigemoto et?al., 1996; Sylwestrak and Ghosh, 2012). Furthermore, the structural and functional properties of synapses made by identical pre- and postsynaptic cell types are also widely different (e.g., synapses among cerebellar Hexachlorophene interneurons [INs]; Pulido et?al., 2015; or among hippocampal CA3 pyramidal cells [PCs]; Holderith et?al., 2012). In these cases, population-level analysis of the functional properties and molecular content of synapses are inadequate; only individual synapse-based methods are suitable. What are the currently available high-resolution localization methods? The most widely used method is usually pre-embedding, light microscopy (LM) immunohistochemistry on solid (10C100?m) sections. This method, however, is highly nonquantitative due to the differential diffusion of immunoreagents into the depth of the tissue. Only diffusion-free methods permit quantitative comparisons in the reaction strength of different subcellular elements. Freeze-fracture imitation immunolabeling (FRL; Fujimoto et?al., 1996; Rash et?al., 1998) has been widely used to localize transmembrane proteins at high resolution. This method is usually diffusion free, is usually linear, and has an outstanding sensitivity (Masugi-Tokita and Shigemoto, 2007); but, due to the random fracturing of the frozen tissue, so far none could use this method to study individual, functionally characterized synapses. Post-embedding immunolocalization on thin (100?nm) resin-embedded tissue sections is also diffusion free, is quantitative, and allows the visualization of antigens embedded in dense protein matrices (e.g., receptors embedded in the postsynaptic density [PSD]; Nusser, 1999; Ottersen and Landsend, 1997; Phend et?al., 1995). Experts of previous studies (Collman et?al., 2015; Micheva and Bruchez, 2012; Micheva and Smith, 2007) have visualized the post-embedding reactions with fluorescent-dye-coupled secondary antibodies (sAbs), Hexachlorophene imaged them at the LM level, and performed the reactions on serial sections for post hoc reconstruction of the volume of the tissue (method Hexachlorophene called array tomography [AT]). They also demonstrated that this Abs can be eluted after a labeling round and Igf2r that the sections can be relabeled, allowing the visualization of dozens of molecules. Probably the major shortcoming of AT was its limited sensitivity, and its application to functionally characterized synapses was challenging (Valenzuela et?al., 2016). Here, we aim to overcome these limitations by optimizing fixation, resin, embedding, etching, retrieval, and elution conditions. We discovered that the highest sensitivity of the reactions was achieved in regular epoxy-resin-embedded tissue following etching and retrieval and also demonstrate the straightforward applicability of our method to functionally characterized synapses. Results Etching Epoxy-Resin-Embedded Ultrathin Sections Dramatically Increases Immunolabeling We performed post-embedding immunoreactions on cerebellar sections embedded into epoxy (Durcupan) or acrylic (LR White and Lowicryl HM20) resins without OsO4 treatment by using standard protocols, and we visualized the reactions with fluorescent-dye-coupled sAbs. According to published results (Micheva and Smith, 2007), we detected punctate neuropil labeling for the soluble NSF attachment proteins receptors (SNARE) complex protein SNAP25 in the cerebellar molecular layer in acrylic-resin-embedded sections, without any detectable transmission on.