Immunocytochemistry for amino acids with post-embeddiag gold is compatible with glutaraldehyde fmtion, osmidon, and embedding in epoxy-based plastics, but h u n o g o l d detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, andlor embedding in acrylic plastics. These make samples more dif5cult to prepare and view and may compmmke structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method opti- IntroductionPost-embedding gold labeling for electron microscopic (EM) immunocytochemistry provides important advantages over preembedding methods, including better localization and structural preservation (Stirling, 1990;Hayat, 1989). It is also suitable for quantitative study, as gold particle density can be directly measured while the problem of variable antibody penetration is avoided. Postembedding immunocytochemistry on standard material (fixed in glutaraldehyde, post-fixed in 0~0 4 , and embedded in epoxy plastics) works well for the study of amino acids (Phend et al., 1992;Van den Pol et al., 1990;Ottersen, 1989; Somogyi and Soltbz, 1986;. However, many larger molecules may completely lose immunoreactivity, or label with only low sensitivity and high background. A number of modifications have been introduced, but each has shortcomings (see Discussion). We report here a technique that preserves immunoreactivity with only minimal associated dkficulties.os04 is a highly reactive fixative, preserving membranes at least in part by bonding to unsaturated lipids (Hayat, 1989). Unfortunately, os04 may also react with tissue antigens, making it un-' Supported by NINDS awards #12440 and 16164 (AR) and #29879 suitable for optimal immunocytochemistry (Stirling, 1989;Bendayan et al., 1987;Pearse, 1980). In an effort to find ways to avoid this problem, we considered other agents reputed to preserve membranes. En bloc staining with uranyl acetate (UAc), often used after os04 to enhance specimen contrast, may also act as a fixative to help preserve structure (Silva et al., 1971;Terzakis, 1968). Unlike 0~0 4 , UAc is not highly reactive; its fixative effect appears to arise from electrostatic interactions between the uranyl cation and tissue elements. Perhaps for this reason, UAc appears to have little or no adverse effect on immunostaining (Stirling, 1992;Berryman and Rodewald, 1990;Erickson et al., 1987). Tissue preservation via non-covalent interactions can be achieved by tannic acid (TA, a mixture of polyglycol anions), which has been used in histology for many years as mordant and adjuvant fixative, both for light (reviewed by Chaplin, 1985) and electron microscopy (Sannes et al., 1978; Kalina and Pease, 1977; LaFountain et al., 1977;Simionescu and Simionescu, 1976;Wagner, 1976; van Deurs, 1971;Mizuhira and Futaesaku, 1971). After embedding in ...

Synapses of the mammalian CNS are diverse in size, structure, molecular composition, and function. Synapses in their myriad variations are fundamental to neural circuit development, homeostasis, plasticity, and memory storage. Unfortunately, quantitative analysis and mapping of the brain's heterogeneous synapse populations has been limited by the lack of adequate single-synapse measurement methods. Electron microscopy (EM) is the definitive means to recognize and measure individual synaptic contacts, but EM has only limited abilities to measure the molecular composition of synapses. This report describes conjugate array tomography (AT), a volumetric imaging method that integrates immunofluorescence and EM imaging modalities in voxel-conjugate fashion. We illustrate the use of conjugate AT to advance the proteometric measurement of EM-validated single-synapse analysis in a study of mouse cortex.

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.

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In the presence of netrin, tripartite motif protein 9 (TRIM9) promotes deleted in colorectal cancer (DCC) clustering, but TRIM9-dependent ubiquitination of DCC is reduced. Loss of ubiquitination promotes an interaction between DCC and FAK and FAK activation. FAK activation is required for the progression from SNARE assembly to exocytic vesicle fusion, which supplies membrane material for axon branching.