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  • 99
    Thermo Fisher nanogold conjugated anti rabbit secondary antibody
    Fbxo45 protein expression. A , immunoblot analysis of Fbxo45 in adult mice is shown. B , subcellular distribution of Fbxo45 in the mouse brain is shown. Immunoblot analysis of Fbxo45, Munc13, SNAP25, synaptophysin ( Syph ), synaptotagmin ( Syt ), synaptogyrin ( Syngr ), or PSD-95 in the indicated fractions showed that Fbxo45 was enriched at the synaptic region, including the synaptosomal membrane fraction ( LP1 ) and postsynaptic density ( PSD ). H , homogenate; P1 , nuclear fraction; S1 , crude synaptosomal fraction; S2 , cytosolic synaptosomal fraction; P2 , crude synaptosomal pellet fraction; LP2 , synaptosomal vesicle fraction; SPM , synaptic plasma membrane fraction. C , immunocytochemistry of primary cultured rat hippocampal neurons expressing Fbxo45 is shown. Shown are Fbxo45 ( green ), VGlut1 ( red ), and PSD-95 ( purple ) ( upper panels ). Magnifications of the boxed areas in the upper panels are shown in the lower panels. Arrowheads indicate synapses, at which VGlut1 and PSD-95 were colocalized. Scale bars : 20 μm in the upper panels , 5 μm in the lower panels ). D , shown is immunoelectron microscopy of endogenous Fbxo45. The lower panel shows a magnified image of the boxed area in the upper panel. S , synapse; Pre , presynaptic terminal; Post , postsynaptic terminal. Scale bars : 0.5 μm in the upper panel , 0.2 μm in the lower panel. E , localization of <t>nanogold</t> particles was quantitatively evaluated on each indicated structures (count/μm 2 ). Asterisk , unpaired t test, p
    Nanogold Conjugated Anti Rabbit Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nanogold fiducial bsa solution
    Fbxo45 protein expression. A , immunoblot analysis of Fbxo45 in adult mice is shown. B , subcellular distribution of Fbxo45 in the mouse brain is shown. Immunoblot analysis of Fbxo45, Munc13, SNAP25, synaptophysin ( Syph ), synaptotagmin ( Syt ), synaptogyrin ( Syngr ), or PSD-95 in the indicated fractions showed that Fbxo45 was enriched at the synaptic region, including the synaptosomal membrane fraction ( LP1 ) and postsynaptic density ( PSD ). H , homogenate; P1 , nuclear fraction; S1 , crude synaptosomal fraction; S2 , cytosolic synaptosomal fraction; P2 , crude synaptosomal pellet fraction; LP2 , synaptosomal vesicle fraction; SPM , synaptic plasma membrane fraction. C , immunocytochemistry of primary cultured rat hippocampal neurons expressing Fbxo45 is shown. Shown are Fbxo45 ( green ), VGlut1 ( red ), and PSD-95 ( purple ) ( upper panels ). Magnifications of the boxed areas in the upper panels are shown in the lower panels. Arrowheads indicate synapses, at which VGlut1 and PSD-95 were colocalized. Scale bars : 20 μm in the upper panels , 5 μm in the lower panels ). D , shown is immunoelectron microscopy of endogenous Fbxo45. The lower panel shows a magnified image of the boxed area in the upper panel. S , synapse; Pre , presynaptic terminal; Post , postsynaptic terminal. Scale bars : 0.5 μm in the upper panel , 0.2 μm in the lower panel. E , localization of <t>nanogold</t> particles was quantitatively evaluated on each indicated structures (count/μm 2 ). Asterisk , unpaired t test, p
    Nanogold Fiducial Bsa Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Nanoprobes nanogold
    Quantitation of <t>Nanogold-labeled</t> structures. Wild-type and tlg2Δ spheroplasts were incubated with positively charged Nanogold on ice for 5 min and then shifted to 15°C for various times up to 60 min. The cells were fixed, dehydrated, and embedded. Thin sections were generated and enhanced with HQ Silver and visualized in the electron microscope. Labeled vesicles, early endosomes, and late endosomes were identified on sections and quantified. The vesicle quantitation is shown in A, the early and late endosome quantitation in B.
    Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 91/100, based on 665 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Nanoprobes ni nta nanogold
    Three-dimensional location of the cytoplasmic N terminus of His 6 -Wzc identified by <t>Ni-NTA-nanogold</t> labeling
    Ni Nta Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 89/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanoprobes monomaleimido nanogold
    Preparation of Au-Fc. (a) Ribbon diagram of the structure of Fc (pdb code 1I1c) with a gold sphere (drawn to scale) representing a 1.4 nm <t>monomaleimido</t> <t>Nanogold</t> covalently attached to a reduced cysteine in the Fc hinge region. One Nanogold is depicted, based on the calculated Nanogold/Fc ratios obtained in most labeling reactions (0.8 - 1.1). The Nanogold cluster bound in a region that is distant from the FcRn binding site (indicated on each Fc chain with a bracket). (b) S75 Superdex gel filtration profile following incubation of reduced Fc with 1.4 nm Nanogold. (c) 10% SDS-PAGE analysis of non-reduced and unboiled unlabeled Fc (lane 1) and Au-Fc (lane 2). The majority of Fc protein migrated at a higher apparent molecular weight in the Au-Fc sample, demonstrating covalent attachment of 1.4 nm Nanogold. (d) Confocal images (∼5 μm below the apical surface) of FcRn-expressing MDCK cells after Au-Fc uptake (bar = 10 μm). Filter-grown monolayers were incubated with ∼1 μM Au-Fc for one hour at pH 6 and processed for immunofluorescence using antibodies against FcRn (green; left panel) and Fc (red; middle panel) as described in the Supplementary Methods. The merged image (right panel) shows regions of colocalization as yellow. The nearly equimolar ratio of gold to protein in our Au-Fc samples (see Methods) suggested that most or all of the Fc detected by immunofluorescence contained gold. Untransfected MDCK cells showed only background levels of fluorescence when subjected to the same incubation and staining protocols (data not shown).
    Monomaleimido Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 89/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Nanoprobes nanogold conjugated secondary antibody
    Correlative light and electron microscopy imaging of a 2P uncaging modulated dendritic spine. (Left panel) Workflow and estimated cost time (A1-A2) 1 x and 5 x 2P images of a GFP positive CA1 pyramidal neuron in organotypic hippocampal slice selected for glutamate uncaging. Scale bar: A1, 20 µm, A2, 5 µm. (A3-A4) 25 x 2P images of a dendritic spine before (A3) and after (A4) 2P glutamate uncaging stimulation. Red dot in (A3) shows the uncaging position, and red arrow in (A4) points to the enlarged dendritic spine. Scale bar: 1 µm. (B1) 25x 2p image of the uncaged spine after fixation. Arrow points at the target spine. Scale bar: 1 µm. (B2) Laser burning fiducial mark is introduced by 2p laser near the target spine after tissue fixation. Scale bar: 1 µm. (B3) A confocal image shows the overview of GFP expression pattern in the whole hippocampal organotypic slice. Target neuron is labeled in red square. Scale bar: 200 µm. (C1) ATUMtome setup for sectioning and collecting ultrathin sections. (C2) Silicon wafer holding aligned Kapton tape containing serial sections. 3 copper grids on the edge of the wafer are used as fiducial markers for alignment. (D1) 2 µm/pixel image taken with SE2 detector shows the outline of two ultrathin sections. Scale bar: 200 µm. (D2) 30 nm/pixel image taken with BSE detector shows tissue structure and the silver enhanced <t>nanogold</t> labeling pattern. Nanogolds are seen as black dots in the image. Scale bar: 50 µm. (D3) 4 nm/pixel high resolution image taken with InlensDuo detector shows details of modulated dendritic spine ultrastructure. Scale bar: 0.5 µm. (D4) 3D reconstruction of the target dendritic spine. Spine head colored in yellow, and PSD colored in red. Scale bar: 200 nm.
    Nanogold Conjugated Secondary Antibody, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 85/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nanoprobes streptavidin nanogold
    Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by <t>streptavidin-nanogold</t> and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.
    Streptavidin Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Nanoprobes anti rabbit nanogold
    Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by <t>streptavidin-nanogold</t> and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.
    Anti Rabbit Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 88/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Microelectrodes Inc nanogold film interdigital microelectrodes
    Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by <t>streptavidin-nanogold</t> and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.
    Nanogold Film Interdigital Microelectrodes, supplied by Microelectrodes Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanoprobes goat anti rabbit igg nanogold
    Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by <t>streptavidin-nanogold</t> and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.
    Goat Anti Rabbit Igg Nanogold, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 85/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanoprobes nanogold solution
    Charged <t>Nanogold-labelling</t> of biofilm matrix components. ( a ) Two-hour biofilms of S. aureus SH1000 were labelled with positively charged Nanogold (PCG), negatively charged Nanogold (NCG), or colloidal gold-conjugated wheat germ agglutinin (WGA-gold). PCG and NCG were used sequentially or, if required, simultaneously, as indicated. After gold enhancement, specimens were observed by ASEM. ( b ) Four-hour biofilms of S. aureus MR10 labelled with PCG, gold-enhanced, and observed by ASEM. A higher magnification image of the white rectangle is shown on the right. The arrow indicates a PCG-positive fibril. ( c ) S. aureus MR10 biofilms were grown in BHIG medium supplemented with, or without, the indicated enzymes for 4 h at 37 °C, labelled with PCG, and observed by ASEM. Arrows mark PCG-positive fibrillar structures. ( d ) Four-hour biofilms of MR10 were labelled with anti-dsDNA mouse IgG primary antibody and colloidal gold-conjugated anti-mouse IgG secondary antibody (anti-dsDNA). The biofilms were subsequently counter-stained with PCG (anti-dsDNA/PCG). A higher magnification image of the white rectangle is shown on the right. Arrowheads and arrows mark linearly aligned colloidal gold particles and PCG-positive fibrillar structures, respectively. Scale bars, 1 μm.
    Nanogold Solution, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nanoprobes nanogold conjugated goat anti rabbit igg
    Charged <t>Nanogold-labelling</t> of biofilm matrix components. ( a ) Two-hour biofilms of S. aureus SH1000 were labelled with positively charged Nanogold (PCG), negatively charged Nanogold (NCG), or colloidal gold-conjugated wheat germ agglutinin (WGA-gold). PCG and NCG were used sequentially or, if required, simultaneously, as indicated. After gold enhancement, specimens were observed by ASEM. ( b ) Four-hour biofilms of S. aureus MR10 labelled with PCG, gold-enhanced, and observed by ASEM. A higher magnification image of the white rectangle is shown on the right. The arrow indicates a PCG-positive fibril. ( c ) S. aureus MR10 biofilms were grown in BHIG medium supplemented with, or without, the indicated enzymes for 4 h at 37 °C, labelled with PCG, and observed by ASEM. Arrows mark PCG-positive fibrillar structures. ( d ) Four-hour biofilms of MR10 were labelled with anti-dsDNA mouse IgG primary antibody and colloidal gold-conjugated anti-mouse IgG secondary antibody (anti-dsDNA). The biofilms were subsequently counter-stained with PCG (anti-dsDNA/PCG). A higher magnification image of the white rectangle is shown on the right. Arrowheads and arrows mark linearly aligned colloidal gold particles and PCG-positive fibrillar structures, respectively. Scale bars, 1 μm.
    Nanogold Conjugated Goat Anti Rabbit Igg, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 91/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Nanoprobes nanogold labeling
    ASEM images by positively charged <t>Nanogold</t> labeling. ( A ) Cells cultured on the ASEM dish were fixed, stained with positively charged Nanogold, and treated with GoldEnhance-EM. 3500× magnification. Scale bar, 5 μm; and ( B ) The cell body was further magnified at 8000× magnification. Scale bar, 2 μm.
    Nanogold Labeling, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 88/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Nanoprobes nanogold conjugated fab fragments
    Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1 ). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the <t>nanogold</t> gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.
    Nanogold Conjugated Fab Fragments, supplied by Nanoprobes, used in various techniques. Bioz Stars score: 88/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Aurion nanogold
    Bacteria in ovarian tissue labeled with <t>Nanogold</t> and silver enhanced for 15 minutes to improve visualization by SEM. Scale bar = 0.5 μm.
    Nanogold, supplied by Aurion, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Nanoprobes goat anti rabbit nanogold
    Immunoelectron microscopic localization of APPL1. HeLa cells were labeled with antibodies to APPL1, followed by a <t>Nanogold-labeled</t> conjugate. Gold particles were visualized by silver enhancement. (A) A low-magnification view with two APPL-positive structures circled in orange, one of which is shown at higher magnification in the inset. (B–F) A gallery of representative structures. Dense labeling is associated with small tubular profiles close to the PM or deeper inside the cell as well as with larger heterogeneous structures. Note that labeling of the PM or CCPs is very low. M, mitochondria. Bars: (A, C, and E) 500 nm; (A [inset], B, D, and F) 200 nm.
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    Nanoprobes nanogold conjugated anti rabbit igg
    Immunoelectron microscopic localization of APPL1. HeLa cells were labeled with antibodies to APPL1, followed by a <t>Nanogold-labeled</t> conjugate. Gold particles were visualized by silver enhancement. (A) A low-magnification view with two APPL-positive structures circled in orange, one of which is shown at higher magnification in the inset. (B–F) A gallery of representative structures. Dense labeling is associated with small tubular profiles close to the PM or deeper inside the cell as well as with larger heterogeneous structures. Note that labeling of the PM or CCPs is very low. M, mitochondria. Bars: (A, C, and E) 500 nm; (A [inset], B, D, and F) 200 nm.
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    Nanoprobes nanogold enhancer
    The filamentous nature of RRs is evident by EM staining and ARL2 and IMPDH2 colocalize to RRs as seen by immunogold staining. HeLa cells were induced with 1 μM MPA for 2 h and processed for EM, as described under Materials and Methods . All scale bars = 200 nm. (A) Longitudinal sections of RRs. The RRs are indicated by an asterisk (*). (B) A zoomed-out image of the RR shown in the far-right image from A. (C) Transverse sections of RRs. Labeling scheme is the same as A. (D) Immuno-EM showing localization of IMPDH2 using IMPDH2 antibody coupled to <t>nanogold</t> particles. The asterisk marks the RR while the black particles indicate the IMPDH2 localization. (E) Same as D except using ARL2 antibody to show localization of ARL2. (F) The fraction of RRs that were associated with the ER or mitochondrial membranes were counted in randomly acquired EM images of the RRs and plotted. N = 45.
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    Image Search Results


    Fbxo45 protein expression. A , immunoblot analysis of Fbxo45 in adult mice is shown. B , subcellular distribution of Fbxo45 in the mouse brain is shown. Immunoblot analysis of Fbxo45, Munc13, SNAP25, synaptophysin ( Syph ), synaptotagmin ( Syt ), synaptogyrin ( Syngr ), or PSD-95 in the indicated fractions showed that Fbxo45 was enriched at the synaptic region, including the synaptosomal membrane fraction ( LP1 ) and postsynaptic density ( PSD ). H , homogenate; P1 , nuclear fraction; S1 , crude synaptosomal fraction; S2 , cytosolic synaptosomal fraction; P2 , crude synaptosomal pellet fraction; LP2 , synaptosomal vesicle fraction; SPM , synaptic plasma membrane fraction. C , immunocytochemistry of primary cultured rat hippocampal neurons expressing Fbxo45 is shown. Shown are Fbxo45 ( green ), VGlut1 ( red ), and PSD-95 ( purple ) ( upper panels ). Magnifications of the boxed areas in the upper panels are shown in the lower panels. Arrowheads indicate synapses, at which VGlut1 and PSD-95 were colocalized. Scale bars : 20 μm in the upper panels , 5 μm in the lower panels ). D , shown is immunoelectron microscopy of endogenous Fbxo45. The lower panel shows a magnified image of the boxed area in the upper panel. S , synapse; Pre , presynaptic terminal; Post , postsynaptic terminal. Scale bars : 0.5 μm in the upper panel , 0.2 μm in the lower panel. E , localization of nanogold particles was quantitatively evaluated on each indicated structures (count/μm 2 ). Asterisk , unpaired t test, p

    Journal: The Journal of Biological Chemistry

    Article Title: Fbxo45, a Novel Ubiquitin Ligase, Regulates Synaptic Activity *

    doi: 10.1074/jbc.M109.046284

    Figure Lengend Snippet: Fbxo45 protein expression. A , immunoblot analysis of Fbxo45 in adult mice is shown. B , subcellular distribution of Fbxo45 in the mouse brain is shown. Immunoblot analysis of Fbxo45, Munc13, SNAP25, synaptophysin ( Syph ), synaptotagmin ( Syt ), synaptogyrin ( Syngr ), or PSD-95 in the indicated fractions showed that Fbxo45 was enriched at the synaptic region, including the synaptosomal membrane fraction ( LP1 ) and postsynaptic density ( PSD ). H , homogenate; P1 , nuclear fraction; S1 , crude synaptosomal fraction; S2 , cytosolic synaptosomal fraction; P2 , crude synaptosomal pellet fraction; LP2 , synaptosomal vesicle fraction; SPM , synaptic plasma membrane fraction. C , immunocytochemistry of primary cultured rat hippocampal neurons expressing Fbxo45 is shown. Shown are Fbxo45 ( green ), VGlut1 ( red ), and PSD-95 ( purple ) ( upper panels ). Magnifications of the boxed areas in the upper panels are shown in the lower panels. Arrowheads indicate synapses, at which VGlut1 and PSD-95 were colocalized. Scale bars : 20 μm in the upper panels , 5 μm in the lower panels ). D , shown is immunoelectron microscopy of endogenous Fbxo45. The lower panel shows a magnified image of the boxed area in the upper panel. S , synapse; Pre , presynaptic terminal; Post , postsynaptic terminal. Scale bars : 0.5 μm in the upper panel , 0.2 μm in the lower panel. E , localization of nanogold particles was quantitatively evaluated on each indicated structures (count/μm 2 ). Asterisk , unpaired t test, p

    Article Snippet: For immunoelectron microscopic analysis, frozen sections were incubated with the anti-Fbxo45 primary antibody (1:500) followed by incubation with a nanogold-conjugated anti-rabbit secondary antibody (1:100; Invitrogen).

    Techniques: Expressing, Mouse Assay, Immunocytochemistry, Cell Culture, Immuno-Electron Microscopy

    Quantitation of Nanogold-labeled structures. Wild-type and tlg2Δ spheroplasts were incubated with positively charged Nanogold on ice for 5 min and then shifted to 15°C for various times up to 60 min. The cells were fixed, dehydrated, and embedded. Thin sections were generated and enhanced with HQ Silver and visualized in the electron microscope. Labeled vesicles, early endosomes, and late endosomes were identified on sections and quantified. The vesicle quantitation is shown in A, the early and late endosome quantitation in B.

    Journal: Molecular Biology of the Cell

    Article Title: A Yeast t-SNARE Involved in Endocytosis

    doi:

    Figure Lengend Snippet: Quantitation of Nanogold-labeled structures. Wild-type and tlg2Δ spheroplasts were incubated with positively charged Nanogold on ice for 5 min and then shifted to 15°C for various times up to 60 min. The cells were fixed, dehydrated, and embedded. Thin sections were generated and enhanced with HQ Silver and visualized in the electron microscope. Labeled vesicles, early endosomes, and late endosomes were identified on sections and quantified. The vesicle quantitation is shown in A, the early and late endosome quantitation in B.

    Article Snippet: One ml was incubated with 5 nmol of positively charged Nanogold (Nanoprobes, Stony Brook, NY) at 0°C for 15 min and then warmed to 15°C or room temperature before fixing by addition of formaldehyde and glutaraldehyde to final concentrations of 3 and 0.2%, respectively.

    Techniques: Quantitation Assay, Labeling, Incubation, Generated, Microscopy

    Internalization of positively charged Nanogold. Wild-type (A and B) and tlg2Δ (C–I) spheroplasts were incubated with positively charged Nanogold on ice for 5 min and then shifted to room temperature for 15 min. The cells were fixed, dehydrated, and embedded. Thin sections were generated and enhanced with HQ Silver and visualized in the electron microscope. Endocytic vesicles found in tlg2Δ cells are labeled with arrows; an early endosome from wild-type cells is labeled with an asterisk, as is a structure from tlg2Δ cells, which resembles an early endosome somewhat. The triangle in A indicates late endosomes. Bar, 200 nm.

    Journal: Molecular Biology of the Cell

    Article Title: A Yeast t-SNARE Involved in Endocytosis

    doi:

    Figure Lengend Snippet: Internalization of positively charged Nanogold. Wild-type (A and B) and tlg2Δ (C–I) spheroplasts were incubated with positively charged Nanogold on ice for 5 min and then shifted to room temperature for 15 min. The cells were fixed, dehydrated, and embedded. Thin sections were generated and enhanced with HQ Silver and visualized in the electron microscope. Endocytic vesicles found in tlg2Δ cells are labeled with arrows; an early endosome from wild-type cells is labeled with an asterisk, as is a structure from tlg2Δ cells, which resembles an early endosome somewhat. The triangle in A indicates late endosomes. Bar, 200 nm.

    Article Snippet: One ml was incubated with 5 nmol of positively charged Nanogold (Nanoprobes, Stony Brook, NY) at 0°C for 15 min and then warmed to 15°C or room temperature before fixing by addition of formaldehyde and glutaraldehyde to final concentrations of 3 and 0.2%, respectively.

    Techniques: Incubation, Generated, Microscopy, Labeling

    TEM images of nanostructures. a Nanotubes and nanofibers formed from compounds A ( left ) and C ( right ), respectively, stained with uranyl acetate. b TEM images of Ni–NTA Nanogold ® particles bound to the hexa-histidine tagged form I RubisCO and associated with either nanotube A ( left ) or nanofiber C ( right ). Images are representative of multiple samples imaged from independent preparations. For better clarity, a close-up view of a single nanostructure is shown to the left of each image

    Journal: Biotechnology for Biofuels

    Article Title: Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO

    doi: 10.1186/s13068-017-0861-6

    Figure Lengend Snippet: TEM images of nanostructures. a Nanotubes and nanofibers formed from compounds A ( left ) and C ( right ), respectively, stained with uranyl acetate. b TEM images of Ni–NTA Nanogold ® particles bound to the hexa-histidine tagged form I RubisCO and associated with either nanotube A ( left ) or nanofiber C ( right ). Images are representative of multiple samples imaged from independent preparations. For better clarity, a close-up view of a single nanostructure is shown to the left of each image

    Article Snippet: Nanogold particles were pre-bound to histidine-tagged proteins by following the procedures described elsewhere (Nanoprobes, Inc.).

    Techniques: Transmission Electron Microscopy, Staining

    High-power micrographs capturing the direct link of MVP (arrowheads) to single microtubules (double arrowheads). Immunoparticles distinctly tag the microtubules in cross-sectioned ( A , B ) as well as in longitudinally sectioned ( C ) dendrites, and likewise label cross-sectioned microtubules in myelinated ( D ) and nonmyelinated preterminal segments ( E ) of axons. Arrows point to microfilament side arms bridging the dendritic and axonal microtubules. The immunocomplexes used here have a presumed maximum linear dimension of about 15 nm, which may translate into a distance of the gold core from the epitope. To demonstrate specificity, the micrographs illustrate material labeled with sequential enhancement of nanogold (see Methods). mit, mitochondrion; ml-ax, myelinated axon; mvb, multivesicular body. Scale bars: ( A , D ) 200 nm; ( B , C , E ) 50 nm.

    Journal: Cerebral Cortex (New York, NY)

    Article Title: Major Vault Protein is Expressed along the Nucleus-Neurite Axis and Associates with mRNAs in Cortical Neurons

    doi: 10.1093/cercor/bhn203

    Figure Lengend Snippet: High-power micrographs capturing the direct link of MVP (arrowheads) to single microtubules (double arrowheads). Immunoparticles distinctly tag the microtubules in cross-sectioned ( A , B ) as well as in longitudinally sectioned ( C ) dendrites, and likewise label cross-sectioned microtubules in myelinated ( D ) and nonmyelinated preterminal segments ( E ) of axons. Arrows point to microfilament side arms bridging the dendritic and axonal microtubules. The immunocomplexes used here have a presumed maximum linear dimension of about 15 nm, which may translate into a distance of the gold core from the epitope. To demonstrate specificity, the micrographs illustrate material labeled with sequential enhancement of nanogold (see Methods). mit, mitochondrion; ml-ax, myelinated axon; mvb, multivesicular body. Scale bars: ( A , D ) 200 nm; ( B , C , E ) 50 nm.

    Article Snippet: Anti-MVP was complexed with nanogold-Fab′ but the signal was enhanced with gold (GoldEnhance; Nanoprobes).

    Techniques: Labeling

    HDL readily incorporates with miRNAs in vitro and in vivo a ) TEM image of HDL + Nanogold-labeled miRNA (miR-223-Au) unenhanced (Top); HDL + Nanogold-labeled miRNA (miR-223-Au) gold enhanced (Au e ) (2 min) (Bottom). Scale bars = 100 nm. b .) FPLC separation of radiolabeled HDL ( 3 H-cholesterol) -miRNA ( 32 P-miR-125a) complexes. Red line, HDL + 32 P-miR-125a (37°C reaction); blue line, HDL + 32 P-miR-125a (20°C reaction); purple line, 32 P-miR-125a alone; black dash line, 3 H-HDL + cold miR-125a. Light shading indicates HDL complex zone, dark shading indicates uncomplexed free radiolabeled 32 P-miR-125a and 3 H-HDL zone. Colored arrows indicate peak associations, red (37°C), blue (20°C), purple (no HDL). S200 Column. c .) Quantification of HDL-miR-223 incorporation. hsa-miR-223 (ng ml −1 ) levels post-HDL-IP, miR-223, positive control; native human HDL; native human HDL + miR-223; reconstituted HDL (rHDL); rHDL + miR-223. n=2 d .) Spearman non-parametric correlation between WT mouse and normal human HDL profiles. R=0.68, P

    Journal: Nature cell biology

    Article Title: MicroRNAs are Transported in Plasma and Delivered to Recipient Cells by High-Density Lipoproteins

    doi: 10.1038/ncb2210

    Figure Lengend Snippet: HDL readily incorporates with miRNAs in vitro and in vivo a ) TEM image of HDL + Nanogold-labeled miRNA (miR-223-Au) unenhanced (Top); HDL + Nanogold-labeled miRNA (miR-223-Au) gold enhanced (Au e ) (2 min) (Bottom). Scale bars = 100 nm. b .) FPLC separation of radiolabeled HDL ( 3 H-cholesterol) -miRNA ( 32 P-miR-125a) complexes. Red line, HDL + 32 P-miR-125a (37°C reaction); blue line, HDL + 32 P-miR-125a (20°C reaction); purple line, 32 P-miR-125a alone; black dash line, 3 H-HDL + cold miR-125a. Light shading indicates HDL complex zone, dark shading indicates uncomplexed free radiolabeled 32 P-miR-125a and 3 H-HDL zone. Colored arrows indicate peak associations, red (37°C), blue (20°C), purple (no HDL). S200 Column. c .) Quantification of HDL-miR-223 incorporation. hsa-miR-223 (ng ml −1 ) levels post-HDL-IP, miR-223, positive control; native human HDL; native human HDL + miR-223; reconstituted HDL (rHDL); rHDL + miR-223. n=2 d .) Spearman non-parametric correlation between WT mouse and normal human HDL profiles. R=0.68, P

    Article Snippet: Prior to TEM, miRNA was labeled with 1.4 nm monoamino Nanogold (Nanoprobes) according to the manufacturer’s protocol.

    Techniques: In Vitro, In Vivo, Transmission Electron Microscopy, Labeling, Fast Protein Liquid Chromatography, Positive Control

    Three-dimensional location of the cytoplasmic N terminus of His 6 -Wzc identified by Ni-NTA-nanogold labeling

    Journal:

    Article Title: Periplasmic Protein-Protein Contacts in the Inner Membrane Protein Wzc Form a Tetrameric Complex Required for the Assembly of Escherichia coli Group 1 Capsules*

    doi: 10.1074/jbc.M508078200

    Figure Lengend Snippet: Three-dimensional location of the cytoplasmic N terminus of His 6 -Wzc identified by Ni-NTA-nanogold labeling

    Article Snippet: Using Ni-NTA-nanogold to label oligomeric complexes may also aid single particle averaging, because the prominent electron density acts as a “target” for two-dimensional alignment algorithms ( ). shows a montage of cryo-negatively stained Wzc particles, which have been incubated with Ni-NTA-nanogold.

    Techniques: Labeling

    ATPase activity of CydDC in the presence of lipid. a , ATPase activity of C-His 6 -CydDC ( Fig. 3 a , peak 2 ) reconstituted into E. coli lipids. The parallel control runs received equimolar (total phosphorus) amounts of EPL small unilamellar vesicles. The rest of the procedure was the same as for detergent-solubilized C-His 6 -CydDC. Each of the data points represents the mean ± S.E. of multiple ( n ≥ 3) protein preparations produced from independent membrane batches. b , an electron micrograph of the C-His 6 -CydDC-EPL proteoliposomes after labeling with 5.0 nm Ni-NTA Nanogold. The sample was stained in 2% phosphotungstic acid. Scale bar = 5 nm.

    Journal: The Journal of Biological Chemistry

    Article Title: Structure and Function of the Bacterial Heterodimeric ABC Transporter CydDC

    doi: 10.1074/jbc.M114.590414

    Figure Lengend Snippet: ATPase activity of CydDC in the presence of lipid. a , ATPase activity of C-His 6 -CydDC ( Fig. 3 a , peak 2 ) reconstituted into E. coli lipids. The parallel control runs received equimolar (total phosphorus) amounts of EPL small unilamellar vesicles. The rest of the procedure was the same as for detergent-solubilized C-His 6 -CydDC. Each of the data points represents the mean ± S.E. of multiple ( n ≥ 3) protein preparations produced from independent membrane batches. b , an electron micrograph of the C-His 6 -CydDC-EPL proteoliposomes after labeling with 5.0 nm Ni-NTA Nanogold. The sample was stained in 2% phosphotungstic acid. Scale bar = 5 nm.

    Article Snippet: Labeling with Ni-NTA-Nanogold Purified C-His6 -CydDC was mixed with 5 nm Ni-NTA Nanogold particles (Nanoprobes, Yaphank, NY) at a molar ratio of 2:1 (final concentrations of 56 and 28 μm , respectively) in 20 mm Tris-HCl (pH 7.6), 20% glycerol, 150 mm NaCl, and 0.02% DDM and incubated briefly at room temperature.

    Techniques: Activity Assay, Produced, Labeling, Staining

    C-His 6 -CydDC gel filtration chromatography. a , top panel , elution profile for the affinity-purified C-His 6 -CydDC from three Superdex200 10/300 GL columns connected in tandem. The solid line represents A 280 nm . ○ represent heme absorption ( A 410 nm ). Peak fitting for the A 410 nm curve was performed using a three-parameter Gaussian. Bottom panel , analysis of the peak fractions by SDS gel electrophoresis. Conditions were as described in the legend for Fig. 2 . b , electron micrographs showing the C-His 6 -CydDC particles (stained with 2% phosphotungstic acid) for the peak 2 and 3 fractions. Insets show equivalent samples labeled with Ni-NTA Nanogold. c , CD spectra for the peak 2 and 3 fractions of C-His 6 -CydDC. The spectrum for the CydDC peak 2 overlaps with data points for peak 2 plus 1 μ m and 100 n m hemin, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Structure and Function of the Bacterial Heterodimeric ABC Transporter CydDC

    doi: 10.1074/jbc.M114.590414

    Figure Lengend Snippet: C-His 6 -CydDC gel filtration chromatography. a , top panel , elution profile for the affinity-purified C-His 6 -CydDC from three Superdex200 10/300 GL columns connected in tandem. The solid line represents A 280 nm . ○ represent heme absorption ( A 410 nm ). Peak fitting for the A 410 nm curve was performed using a three-parameter Gaussian. Bottom panel , analysis of the peak fractions by SDS gel electrophoresis. Conditions were as described in the legend for Fig. 2 . b , electron micrographs showing the C-His 6 -CydDC particles (stained with 2% phosphotungstic acid) for the peak 2 and 3 fractions. Insets show equivalent samples labeled with Ni-NTA Nanogold. c , CD spectra for the peak 2 and 3 fractions of C-His 6 -CydDC. The spectrum for the CydDC peak 2 overlaps with data points for peak 2 plus 1 μ m and 100 n m hemin, respectively.

    Article Snippet: Labeling with Ni-NTA-Nanogold Purified C-His6 -CydDC was mixed with 5 nm Ni-NTA Nanogold particles (Nanoprobes, Yaphank, NY) at a molar ratio of 2:1 (final concentrations of 56 and 28 μm , respectively) in 20 mm Tris-HCl (pH 7.6), 20% glycerol, 150 mm NaCl, and 0.02% DDM and incubated briefly at room temperature.

    Techniques: Filtration, Chromatography, Affinity Purification, SDS-Gel, Electrophoresis, Staining, Labeling

    Preparation of Au-Fc. (a) Ribbon diagram of the structure of Fc (pdb code 1I1c) with a gold sphere (drawn to scale) representing a 1.4 nm monomaleimido Nanogold covalently attached to a reduced cysteine in the Fc hinge region. One Nanogold is depicted, based on the calculated Nanogold/Fc ratios obtained in most labeling reactions (0.8 - 1.1). The Nanogold cluster bound in a region that is distant from the FcRn binding site (indicated on each Fc chain with a bracket). (b) S75 Superdex gel filtration profile following incubation of reduced Fc with 1.4 nm Nanogold. (c) 10% SDS-PAGE analysis of non-reduced and unboiled unlabeled Fc (lane 1) and Au-Fc (lane 2). The majority of Fc protein migrated at a higher apparent molecular weight in the Au-Fc sample, demonstrating covalent attachment of 1.4 nm Nanogold. (d) Confocal images (∼5 μm below the apical surface) of FcRn-expressing MDCK cells after Au-Fc uptake (bar = 10 μm). Filter-grown monolayers were incubated with ∼1 μM Au-Fc for one hour at pH 6 and processed for immunofluorescence using antibodies against FcRn (green; left panel) and Fc (red; middle panel) as described in the Supplementary Methods. The merged image (right panel) shows regions of colocalization as yellow. The nearly equimolar ratio of gold to protein in our Au-Fc samples (see Methods) suggested that most or all of the Fc detected by immunofluorescence contained gold. Untransfected MDCK cells showed only background levels of fluorescence when subjected to the same incubation and staining protocols (data not shown).

    Journal: Journal of structural biology

    Article Title: A Freeze Substitution Fixation-Based Gold Enlarging Technique for EM Studies of Endocytosed Nanogold-Labeled Molecules

    doi: 10.1016/j.jsb.2007.07.004

    Figure Lengend Snippet: Preparation of Au-Fc. (a) Ribbon diagram of the structure of Fc (pdb code 1I1c) with a gold sphere (drawn to scale) representing a 1.4 nm monomaleimido Nanogold covalently attached to a reduced cysteine in the Fc hinge region. One Nanogold is depicted, based on the calculated Nanogold/Fc ratios obtained in most labeling reactions (0.8 - 1.1). The Nanogold cluster bound in a region that is distant from the FcRn binding site (indicated on each Fc chain with a bracket). (b) S75 Superdex gel filtration profile following incubation of reduced Fc with 1.4 nm Nanogold. (c) 10% SDS-PAGE analysis of non-reduced and unboiled unlabeled Fc (lane 1) and Au-Fc (lane 2). The majority of Fc protein migrated at a higher apparent molecular weight in the Au-Fc sample, demonstrating covalent attachment of 1.4 nm Nanogold. (d) Confocal images (∼5 μm below the apical surface) of FcRn-expressing MDCK cells after Au-Fc uptake (bar = 10 μm). Filter-grown monolayers were incubated with ∼1 μM Au-Fc for one hour at pH 6 and processed for immunofluorescence using antibodies against FcRn (green; left panel) and Fc (red; middle panel) as described in the Supplementary Methods. The merged image (right panel) shows regions of colocalization as yellow. The nearly equimolar ratio of gold to protein in our Au-Fc samples (see Methods) suggested that most or all of the Fc detected by immunofluorescence contained gold. Untransfected MDCK cells showed only background levels of fluorescence when subjected to the same incubation and staining protocols (data not shown).

    Article Snippet: After concentration, the reduced Fc was labeled with 1.4 nm monomaleimido Nanogold® (Nanoprobes, Inc.), which reacts specifically with reduced sulfhydryls , following the manufacturer’s protocol for labeling IgG.

    Techniques: Labeling, Binding Assay, Filtration, Incubation, SDS Page, Molecular Weight, Expressing, Immunofluorescence, Fluorescence, Staining

    Correlative light and electron microscopy imaging of a 2P uncaging modulated dendritic spine. (Left panel) Workflow and estimated cost time (A1-A2) 1 x and 5 x 2P images of a GFP positive CA1 pyramidal neuron in organotypic hippocampal slice selected for glutamate uncaging. Scale bar: A1, 20 µm, A2, 5 µm. (A3-A4) 25 x 2P images of a dendritic spine before (A3) and after (A4) 2P glutamate uncaging stimulation. Red dot in (A3) shows the uncaging position, and red arrow in (A4) points to the enlarged dendritic spine. Scale bar: 1 µm. (B1) 25x 2p image of the uncaged spine after fixation. Arrow points at the target spine. Scale bar: 1 µm. (B2) Laser burning fiducial mark is introduced by 2p laser near the target spine after tissue fixation. Scale bar: 1 µm. (B3) A confocal image shows the overview of GFP expression pattern in the whole hippocampal organotypic slice. Target neuron is labeled in red square. Scale bar: 200 µm. (C1) ATUMtome setup for sectioning and collecting ultrathin sections. (C2) Silicon wafer holding aligned Kapton tape containing serial sections. 3 copper grids on the edge of the wafer are used as fiducial markers for alignment. (D1) 2 µm/pixel image taken with SE2 detector shows the outline of two ultrathin sections. Scale bar: 200 µm. (D2) 30 nm/pixel image taken with BSE detector shows tissue structure and the silver enhanced nanogold labeling pattern. Nanogolds are seen as black dots in the image. Scale bar: 50 µm. (D3) 4 nm/pixel high resolution image taken with InlensDuo detector shows details of modulated dendritic spine ultrastructure. Scale bar: 0.5 µm. (D4) 3D reconstruction of the target dendritic spine. Spine head colored in yellow, and PSD colored in red. Scale bar: 200 nm.

    Journal: bioRxiv

    Article Title: Rapid Ultrastructural Changes of PSD and Extrasynaptic Axon-spine Interface Membrane during LTP Induced in Single Dendritic Spine

    doi: 10.1101/840629

    Figure Lengend Snippet: Correlative light and electron microscopy imaging of a 2P uncaging modulated dendritic spine. (Left panel) Workflow and estimated cost time (A1-A2) 1 x and 5 x 2P images of a GFP positive CA1 pyramidal neuron in organotypic hippocampal slice selected for glutamate uncaging. Scale bar: A1, 20 µm, A2, 5 µm. (A3-A4) 25 x 2P images of a dendritic spine before (A3) and after (A4) 2P glutamate uncaging stimulation. Red dot in (A3) shows the uncaging position, and red arrow in (A4) points to the enlarged dendritic spine. Scale bar: 1 µm. (B1) 25x 2p image of the uncaged spine after fixation. Arrow points at the target spine. Scale bar: 1 µm. (B2) Laser burning fiducial mark is introduced by 2p laser near the target spine after tissue fixation. Scale bar: 1 µm. (B3) A confocal image shows the overview of GFP expression pattern in the whole hippocampal organotypic slice. Target neuron is labeled in red square. Scale bar: 200 µm. (C1) ATUMtome setup for sectioning and collecting ultrathin sections. (C2) Silicon wafer holding aligned Kapton tape containing serial sections. 3 copper grids on the edge of the wafer are used as fiducial markers for alignment. (D1) 2 µm/pixel image taken with SE2 detector shows the outline of two ultrathin sections. Scale bar: 200 µm. (D2) 30 nm/pixel image taken with BSE detector shows tissue structure and the silver enhanced nanogold labeling pattern. Nanogolds are seen as black dots in the image. Scale bar: 50 µm. (D3) 4 nm/pixel high resolution image taken with InlensDuo detector shows details of modulated dendritic spine ultrastructure. Scale bar: 0.5 µm. (D4) 3D reconstruction of the target dendritic spine. Spine head colored in yellow, and PSD colored in red. Scale bar: 200 nm.

    Article Snippet: Slices were then blocked with normal goat serum and fish skin gelatin, incubated with anti-GFP primary antibody (0.1 µg/ml, ab6556, Abcam, Cambridge, United Kingdom) for 40 h and nanogold conjugated secondary antibody (1:100, #2003, Nanoprobes, NY) for 16 h. Silver enhancement was then performed with HQ silver enhancement kit (#2012, Nanoprobes, NY) to increase the visibility of gold particles.

    Techniques: Electron Microscopy, Imaging, Expressing, Labeling

    Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by streptavidin-nanogold and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.

    Journal: The EMBO Journal

    Article Title: Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo

    doi: 10.1038/sj.emboj.7601242

    Figure Lengend Snippet: Ultrastructural characterization and thin filament length measurements. Experiments were performed on wt and nebulin KO tibialis cranialis muscle. ( A ) Low magnification micrograph of KO sarcomeres reveals normal structure. ( B ) High magnification micrograph of KO sarcomere. Densitometry scans reveal a continuous reduction of A-band density towards the M-line. ( C ) High magnification micrograph of wt sarcomere. A clear H-zone is present that gives rise to a step-like reduction of A-band density. ( D ) Skinned muscle fibers labelled with phalloidin-biotin, followed by streptavidin-nanogold and silver enhancement. ( E ) Top: Schematic showing how the distance between silver grains and the opposite edge of the nearest Z-disk was measured. Bottom: Histograms of obtained distances are shown for wt (bottom left) and KO superimposed with wt (bottom right). In wt sarcomeres, grain distribution is uniform up to a distance of 1.2 μm. In contrast, in KO tissue, the grain distribution gradually decreases from ∼0.4 to ∼1.2 μm.

    Article Snippet: Fibers were skinned, fixed in 3.7% paraformaldehyde labelled overnight with phalloidin-biotin (Molecular Probes, B7474, biotin-XX phalloidin), followed by streptavidin- nanogold (Nanoprobes, 2016, nanogold streptavidin conjugate), silver enhancement as per instructions with Nanogold HQ silver kitfixing, and embedding ( ).

    Techniques:

    Distribution of nanogold-labeled AChE sites in the synaptic cleft of the mouse endplate. (A,B) Electron micrographs of cross sections through endplates of mouse sternomastoid muscles that had been incubated with Fas-biotin followed by 1.4 nm-nanogold-streptavidin and light silver intensification (details under “Materials and Methods” section). Gold particles (black) are seen over the PC ( A , arrowheads) and PJFs, and are predominantly associated with the basal lamina (BL; B , hollow arrows). Bars, 0.2 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fine Localization of Acetylcholinesterase in the Synaptic Cleft of the Vertebrate Neuromuscular Junction

    doi: 10.3389/fnmol.2018.00123

    Figure Lengend Snippet: Distribution of nanogold-labeled AChE sites in the synaptic cleft of the mouse endplate. (A,B) Electron micrographs of cross sections through endplates of mouse sternomastoid muscles that had been incubated with Fas-biotin followed by 1.4 nm-nanogold-streptavidin and light silver intensification (details under “Materials and Methods” section). Gold particles (black) are seen over the PC ( A , arrowheads) and PJFs, and are predominantly associated with the basal lamina (BL; B , hollow arrows). Bars, 0.2 μm.

    Article Snippet: In additional control, exposed muscles were directly treated with nanogold-streptavidin (or just nanogold particles), omitting the Fas labeling altogether, and then continued as described above.

    Techniques: Labeling, Incubation

    Distribution of gold-labeled AChE sites in the synaptic cleft of the frog NMJ. Frog cutaneous pectoris muscles were incubated with nanogold (1.4 nm) conjugated directly with Fas to label AChE (A,B) , or with biotin-α-bungarotoxin followed by nanogold-streptavidin to label nAChR (C) . (A) Nanogold-Fas conjugate labeled exclusively the NMJs, within both the PC and the PJFs. (B) An enlarged synaptic area (framed in A ) shows the association of the gold-labeled AChE sites with the BL in both the PC and the PJFs. (C) In contrast, labeling of nAChR sites with the nanogold-α-bungarotoxin conjugate is seen to be at the crests and mouths of the PJFs. N, nerve; M, muscle; Arrowheads, labeled AChE in PC; Hollow arrows, labeled AChE in PJF; White arrowhead in (C) , labeled nAChR (on postsynaptic membrane). Bars, 0.2 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Fine Localization of Acetylcholinesterase in the Synaptic Cleft of the Vertebrate Neuromuscular Junction

    doi: 10.3389/fnmol.2018.00123

    Figure Lengend Snippet: Distribution of gold-labeled AChE sites in the synaptic cleft of the frog NMJ. Frog cutaneous pectoris muscles were incubated with nanogold (1.4 nm) conjugated directly with Fas to label AChE (A,B) , or with biotin-α-bungarotoxin followed by nanogold-streptavidin to label nAChR (C) . (A) Nanogold-Fas conjugate labeled exclusively the NMJs, within both the PC and the PJFs. (B) An enlarged synaptic area (framed in A ) shows the association of the gold-labeled AChE sites with the BL in both the PC and the PJFs. (C) In contrast, labeling of nAChR sites with the nanogold-α-bungarotoxin conjugate is seen to be at the crests and mouths of the PJFs. N, nerve; M, muscle; Arrowheads, labeled AChE in PC; Hollow arrows, labeled AChE in PJF; White arrowhead in (C) , labeled nAChR (on postsynaptic membrane). Bars, 0.2 μm.

    Article Snippet: In additional control, exposed muscles were directly treated with nanogold-streptavidin (or just nanogold particles), omitting the Fas labeling altogether, and then continued as described above.

    Techniques: Labeling, Incubation

    Localization of the SNAP-tagged C-terminus of DRC5 in Chlamydomonas axonemes. (A-D) Tomographic slices of the averaged 96-nm axonemal repeats from wild type (A and B) and sup-pf4;DRC5-C-SNAP (C and D) viewed in cross-sectional (A and C) and longitudinal views (B and D). White lines indicate the locations of the slices in the respective panels. Note the additional electron density (yellow arrowheads in D) corresponding to the streptavidin-nanogold marking the location of the C-terminus of DRC5 near the center of the nexin-dynein regulatory complex (N-DRC) linker. Other labels: At, A-tubule; Bt, B-tubule; IDA, inner dynein arm; a–e and g, inner dynein arm isoforms; I1α/ I1β/ ICLC, α- and β-heavy chain, and intermediate-light chain complex of I1 dynein; L1, L1 protrusion connecting between the linker and dynein g; ODA, outer dynein arm. Scale bar: 10 nm.

    Journal: bioRxiv

    Article Title: Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

    doi: 10.1101/684316

    Figure Lengend Snippet: Localization of the SNAP-tagged C-terminus of DRC5 in Chlamydomonas axonemes. (A-D) Tomographic slices of the averaged 96-nm axonemal repeats from wild type (A and B) and sup-pf4;DRC5-C-SNAP (C and D) viewed in cross-sectional (A and C) and longitudinal views (B and D). White lines indicate the locations of the slices in the respective panels. Note the additional electron density (yellow arrowheads in D) corresponding to the streptavidin-nanogold marking the location of the C-terminus of DRC5 near the center of the nexin-dynein regulatory complex (N-DRC) linker. Other labels: At, A-tubule; Bt, B-tubule; IDA, inner dynein arm; a–e and g, inner dynein arm isoforms; I1α/ I1β/ ICLC, α- and β-heavy chain, and intermediate-light chain complex of I1 dynein; L1, L1 protrusion connecting between the linker and dynein g; ODA, outer dynein arm. Scale bar: 10 nm.

    Article Snippet: After adding 5 μl of 80 μg/ml 1.4-nm-sized streptavidin nanogold particles (strep-Au, Nanoprobes, Inc.), the suspension was incubated at 4 °C for 4 h. After adding 800 μl of HMEEK buffer, the labeled axonemes were pelleted by centrifugation at 10,000 g for 10 min at 4 °C and resuspended in 200 μl of HMEEK buffer.

    Techniques:

    Charged Nanogold-labelling of biofilm matrix components. ( a ) Two-hour biofilms of S. aureus SH1000 were labelled with positively charged Nanogold (PCG), negatively charged Nanogold (NCG), or colloidal gold-conjugated wheat germ agglutinin (WGA-gold). PCG and NCG were used sequentially or, if required, simultaneously, as indicated. After gold enhancement, specimens were observed by ASEM. ( b ) Four-hour biofilms of S. aureus MR10 labelled with PCG, gold-enhanced, and observed by ASEM. A higher magnification image of the white rectangle is shown on the right. The arrow indicates a PCG-positive fibril. ( c ) S. aureus MR10 biofilms were grown in BHIG medium supplemented with, or without, the indicated enzymes for 4 h at 37 °C, labelled with PCG, and observed by ASEM. Arrows mark PCG-positive fibrillar structures. ( d ) Four-hour biofilms of MR10 were labelled with anti-dsDNA mouse IgG primary antibody and colloidal gold-conjugated anti-mouse IgG secondary antibody (anti-dsDNA). The biofilms were subsequently counter-stained with PCG (anti-dsDNA/PCG). A higher magnification image of the white rectangle is shown on the right. Arrowheads and arrows mark linearly aligned colloidal gold particles and PCG-positive fibrillar structures, respectively. Scale bars, 1 μm.

    Journal: Scientific Reports

    Article Title: Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

    doi: 10.1038/srep25889

    Figure Lengend Snippet: Charged Nanogold-labelling of biofilm matrix components. ( a ) Two-hour biofilms of S. aureus SH1000 were labelled with positively charged Nanogold (PCG), negatively charged Nanogold (NCG), or colloidal gold-conjugated wheat germ agglutinin (WGA-gold). PCG and NCG were used sequentially or, if required, simultaneously, as indicated. After gold enhancement, specimens were observed by ASEM. ( b ) Four-hour biofilms of S. aureus MR10 labelled with PCG, gold-enhanced, and observed by ASEM. A higher magnification image of the white rectangle is shown on the right. The arrow indicates a PCG-positive fibril. ( c ) S. aureus MR10 biofilms were grown in BHIG medium supplemented with, or without, the indicated enzymes for 4 h at 37 °C, labelled with PCG, and observed by ASEM. Arrows mark PCG-positive fibrillar structures. ( d ) Four-hour biofilms of MR10 were labelled with anti-dsDNA mouse IgG primary antibody and colloidal gold-conjugated anti-mouse IgG secondary antibody (anti-dsDNA). The biofilms were subsequently counter-stained with PCG (anti-dsDNA/PCG). A higher magnification image of the white rectangle is shown on the right. Arrowheads and arrows mark linearly aligned colloidal gold particles and PCG-positive fibrillar structures, respectively. Scale bars, 1 μm.

    Article Snippet: Charged Nanogold labelling For charged Nanogold-labelling, bacteria on the ASEM dish were incubated with 6 μM positively and/or negatively charged 1.4 nm Nanogold solution (Nanoprobes, Stony Brook, NY, USA) for 20 min at room temperature as previously reported .

    Techniques: Whole Genome Amplification, Staining

    Outline of ASEM observation of staphylococcal biofilms in solution. ( a ) Bacteria were grown in an appropriate medium in the removable, 35-mm ASEM dish with SiN x O y film window at the centre of its base. After the indicated incubation time at 37 °C, biofilms were fixed with 1% glutaraldehyde (GA) and 4% paraformaldehyde (PFA) for heavy metal-labelling and charged Nanogold-labelling or only with 4% PFA for immuno-labelling. After the indicated labelling procedures, the solution in the ASEM dish was replaced by a radical scavenger solution (10 mg/ml ascorbic acid or 10 mg/ml glucose). ( b ) Diagram of the ASEM showing the inverted SEM, the detector and the specimen dish, which separates the atmosphere (above) and the column vacuum (below). The electron beam passes through the window and is projected up onto the biofilms immersed in solution, penetrating them to a depth of 2–3 μm. Backscattered electrons (BSE) are captured by a BSE imaging (BEI) detector.

    Journal: Scientific Reports

    Article Title: Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

    doi: 10.1038/srep25889

    Figure Lengend Snippet: Outline of ASEM observation of staphylococcal biofilms in solution. ( a ) Bacteria were grown in an appropriate medium in the removable, 35-mm ASEM dish with SiN x O y film window at the centre of its base. After the indicated incubation time at 37 °C, biofilms were fixed with 1% glutaraldehyde (GA) and 4% paraformaldehyde (PFA) for heavy metal-labelling and charged Nanogold-labelling or only with 4% PFA for immuno-labelling. After the indicated labelling procedures, the solution in the ASEM dish was replaced by a radical scavenger solution (10 mg/ml ascorbic acid or 10 mg/ml glucose). ( b ) Diagram of the ASEM showing the inverted SEM, the detector and the specimen dish, which separates the atmosphere (above) and the column vacuum (below). The electron beam passes through the window and is projected up onto the biofilms immersed in solution, penetrating them to a depth of 2–3 μm. Backscattered electrons (BSE) are captured by a BSE imaging (BEI) detector.

    Article Snippet: Charged Nanogold labelling For charged Nanogold-labelling, bacteria on the ASEM dish were incubated with 6 μM positively and/or negatively charged 1.4 nm Nanogold solution (Nanoprobes, Stony Brook, NY, USA) for 20 min at room temperature as previously reported .

    Techniques: Incubation, Imaging

    Immunogold-labelling to detect matrix components in biofilms. ( a ) Immuno-labelled biofilm cells of the MR23 Δ spa Δ sbi mutant (top), which was used to prevent nonspecific binding of IgGs to Spa and Sbi, and the similarly treated MR23 Δ spa Δ sbi Δ eap mutant (bottom), which was used as a negative control. The same areas are shown in the left (immuno-labelling) and the right (counter staining) panels. ( b,c ) Curli production in the wild type and the Δ csgA strains for 7 days at 25 °C observed by negative stain TEM after air-drying ( b ) and in solution by immuno-labelled ASEM ( c ). Curli were labelled with anti-curli rabbit IgG primary antibody and Nanogold-conjugated secondary antibody for the ASEM. Two typical images of these strains are shown. Scale bars, 1 μm.

    Journal: Scientific Reports

    Article Title: Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

    doi: 10.1038/srep25889

    Figure Lengend Snippet: Immunogold-labelling to detect matrix components in biofilms. ( a ) Immuno-labelled biofilm cells of the MR23 Δ spa Δ sbi mutant (top), which was used to prevent nonspecific binding of IgGs to Spa and Sbi, and the similarly treated MR23 Δ spa Δ sbi Δ eap mutant (bottom), which was used as a negative control. The same areas are shown in the left (immuno-labelling) and the right (counter staining) panels. ( b,c ) Curli production in the wild type and the Δ csgA strains for 7 days at 25 °C observed by negative stain TEM after air-drying ( b ) and in solution by immuno-labelled ASEM ( c ). Curli were labelled with anti-curli rabbit IgG primary antibody and Nanogold-conjugated secondary antibody for the ASEM. Two typical images of these strains are shown. Scale bars, 1 μm.

    Article Snippet: Charged Nanogold labelling For charged Nanogold-labelling, bacteria on the ASEM dish were incubated with 6 μM positively and/or negatively charged 1.4 nm Nanogold solution (Nanoprobes, Stony Brook, NY, USA) for 20 min at room temperature as previously reported .

    Techniques: Mutagenesis, Binding Assay, Negative Control, Staining, Transmission Electron Microscopy

    ASEM images by positively charged Nanogold labeling. ( A ) Cells cultured on the ASEM dish were fixed, stained with positively charged Nanogold, and treated with GoldEnhance-EM. 3500× magnification. Scale bar, 5 μm; and ( B ) The cell body was further magnified at 8000× magnification. Scale bar, 2 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Ultrastructural Analysis of Nanogold-Labeled Cell Surface Microvilli in Liquid by Atmospheric Scanning Electron Microscopy and Their Relevance in Cell Adhesion

    doi: 10.3390/ijms141020809

    Figure Lengend Snippet: ASEM images by positively charged Nanogold labeling. ( A ) Cells cultured on the ASEM dish were fixed, stained with positively charged Nanogold, and treated with GoldEnhance-EM. 3500× magnification. Scale bar, 5 μm; and ( B ) The cell body was further magnified at 8000× magnification. Scale bar, 2 μm.

    Article Snippet: The cells were untreated or treated with 10 μM cytochalasin D for 1 h and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 10 min. For detecting CD44 on the cell surface, the cells were incubated with 1% skim milk/PBS for 30 min, with IM7.8.1 antibody for 1 h, and then with Fab’ fragment of Nanogold- and Alexa Fluor 488-conjugated goat anti-rat IgG for 30 min. After fixation with 1% glutaraldehyde for 15 min at room temperature, the Nanogold signal was enhanced using GoldEnhance-EM at room temperature for 5 min. For positively charged Nanogold labeling, glutaraldehyde-fixed cells were incubated with 3 μM positively charged Nanogold solution (Nanoprobes) for 20 min at room temperature.

    Techniques: Labeling, Cell Culture, Staining

    Distribution of CD44 on the plasma membrane, visualized by correlative optical and electron microscopy using ASEM. BW5147 T lymphocytes were labeled with an anti-CD44 monoclonal antibody IM7.8.1, and further with a secondary antibody conjugated both with Alexa Fluor 488 and Nanogold. ( A ) Fluorescence optical microscopy (OM) image; and ( B ) Electron microscopy (EM) image of the same cells after gold enhancement. Signals were clearly observed on the cell body as well as on the microvilli. ASEM images were captured at 3500× magnification. Scale bar represents 5 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Ultrastructural Analysis of Nanogold-Labeled Cell Surface Microvilli in Liquid by Atmospheric Scanning Electron Microscopy and Their Relevance in Cell Adhesion

    doi: 10.3390/ijms141020809

    Figure Lengend Snippet: Distribution of CD44 on the plasma membrane, visualized by correlative optical and electron microscopy using ASEM. BW5147 T lymphocytes were labeled with an anti-CD44 monoclonal antibody IM7.8.1, and further with a secondary antibody conjugated both with Alexa Fluor 488 and Nanogold. ( A ) Fluorescence optical microscopy (OM) image; and ( B ) Electron microscopy (EM) image of the same cells after gold enhancement. Signals were clearly observed on the cell body as well as on the microvilli. ASEM images were captured at 3500× magnification. Scale bar represents 5 μm.

    Article Snippet: The cells were untreated or treated with 10 μM cytochalasin D for 1 h and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 10 min. For detecting CD44 on the cell surface, the cells were incubated with 1% skim milk/PBS for 30 min, with IM7.8.1 antibody for 1 h, and then with Fab’ fragment of Nanogold- and Alexa Fluor 488-conjugated goat anti-rat IgG for 30 min. After fixation with 1% glutaraldehyde for 15 min at room temperature, the Nanogold signal was enhanced using GoldEnhance-EM at room temperature for 5 min. For positively charged Nanogold labeling, glutaraldehyde-fixed cells were incubated with 3 μM positively charged Nanogold solution (Nanoprobes) for 20 min at room temperature.

    Techniques: Electron Microscopy, Labeling, Fluorescence, Microscopy

    Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1 ). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.

    Journal: The Journal of Cell Biology

    Article Title: Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae

    doi: 10.1083/jcb.200108073

    Figure Lengend Snippet: Dynamic behavior of VSVG-GFP during intra-Golgi transport. Cell were transfected with VSVG–GFP, placed on glass bottom microwell dishes with coordinated grids, subjected to the small-pulse protocol, and studied, after releasing the 15°C block, by laser scanning confocal microscope and time-lapse analysis. (A) 4 min after the shift, the Golgi spots containing VSVG–GFP in the central Golgi area were masked by the high ER background. (b and c) Repeated bleaching of the whole cell (except the Golgi area, delineated) removed the ER background and made the spotty pattern of the VSVG in the Golgi zone more evident. (d and e) Half of the Golgi area was bleached and observed 1 min (D) and 5 min (E) after bleaching. No fluorescence recovery was observed. (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200108073/DC1 ). (f and g) This cell was fixed 7 min after releasing the 15°C block and prepared for correlative video light EM using the nanogold gold enhancement method. The region at the center of the white square in (F) was analyzed (it corresponds to the square is the area enlarged in G). As can be seen in G, the spot represents a stack containing VSVG–GFP in a central cisterna (large white square) Arrowheads indicate nuclear pores. (H–M) Cells were treated as for the experiment in panels B and C and observed at 4 min; (H) Image before bleaching; (I) 7 min; (L) 11 min after releasing the temperature block. At 11 min (when some of the spots were starting to leave the Golgi area) it was fixed an stained for TGN46 (red) and VSVG (green) (M). Many of the spots colocalize with the ribbon, whereas others are probably moving out. Bar: (A–E and H–M) 15 μm; (F) 8 μm; (G) 300 nm.

    Article Snippet: Nanogold-conjugated Fab fragments of anti–rabbit IgG and Gold Enhancer were from Nanoprobes (Yaphank).

    Techniques: Transfection, Blocking Assay, Microscopy, Fluorescence, Staining

    VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.

    Journal: The Journal of Cell Biology

    Article Title: Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae

    doi: 10.1083/jcb.200108073

    Figure Lengend Snippet: VSVG and ssHRP are excluded from Golgi vesicles during transit through the Golgi complex. Human fibroblasts (a–c) and COS-7 cells (d–f) were subjected to the ER accumulation–chase protocol (D) or the large (C) or the intermediate (A and B) pulse protocols, fixed 7 min after the release of the temperature block (A–D), and then processed for labeling. In the experiments in (A) and (B) they were labeled for VSVG by the cryo-immunogold technique, in C by the nanogold gold enhance technique, and in D (which shows a tangential thick section), by the preembedding immunoperoxidase method. Also in A, the GM130 protein is labeled (small particles). (e and f) For ssHRP experiments, COS-7 cells were transfected with ssHRP, fixed at steady state, and then processed for detection of HRP. (E) Perpendicular and (F) tangential section. Irrespective of the labeling and sectioning technique, the round (vesicular) profiles (arrows) are almost always devoid of cargo, whereas cisternae are labeled. Bar: (A, D, and E) 110 nm; (B) 120 nm; (C) 90 nm; (F) 200 nm.

    Article Snippet: Nanogold-conjugated Fab fragments of anti–rabbit IgG and Gold Enhancer were from Nanoprobes (Yaphank).

    Techniques: Blocking Assay, Labeling, Transfection

    Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.

    Journal: The Journal of Cell Biology

    Article Title: Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae

    doi: 10.1083/jcb.200108073

    Figure Lengend Snippet: Human fibroblasts can express and transport both PC-I aggregates and VSVG through the Golgi complex. Human fibroblasts were stimulated to synthesize PC-I and infected with ts045-VSV. After accumulation of both and PC-I and VSVG in the ER at 40°C for 3 h, cells were shifted to 32°C for 9 min and then fixed, permeabilized with saponin, and prepared for immunofluorescence or fixed and prepared for immuno-EM. (A and B) Immunofluorescent double labeling for VSVG (red) and PC-I (green). Both cargoes localize mostly in the Golgi area. The two labeling patterns are very similar. (C) Immuno-EM labeling of PC-I by the preembedding nanogold gold enhancement technique. An aggregate appears as thick cluster of gold particles in a tangential section of a Golgi cisterna. (D) Immuno-EM labeling of VSVG by the same technique. VSVG is distributed throughout the Golgi membranes, including PC-I–containing distensions. Many typical cisternal distensions (*) are seen within the Golgi ribbon (arrows). (E–H) Synchronization protocols. (E) Pulse protocols: cells were kept at 32°C in the presence of AA (50 μg/ml) for 3 h, shifted to 40°C for 3 h (in some experiments 1–2 h), and then shifted to 15°C for 2 h (large pulse), 45 min (intermediate pulse), or 15 min (small pulse), and finally shifted back to 40°C. (F) ER accumulation–chase: cells were kept at 40°C for 3 h in the absence of AA, and then shifted to 32°C in the presence of AA. (G) ER accumulation–pulse: same as for ER accumulation–chase except that cells were shifted back to 40°C after 5 min at 32°C. (H) Exiting wave protocol (Results). Bar: (A and B) 200 nm; (C and D) 2 μm.

    Article Snippet: Nanogold-conjugated Fab fragments of anti–rabbit IgG and Gold Enhancer were from Nanoprobes (Yaphank).

    Techniques: Infection, Immunofluorescence, Labeling

    Bacteria in ovarian tissue labeled with Nanogold and silver enhanced for 15 minutes to improve visualization by SEM. Scale bar = 0.5 μm.

    Journal: Current Protocols in Microbiology

    Article Title: Scanning Electron Microscopy

    doi: 10.1002/9780471729259.mc02b02s25

    Figure Lengend Snippet: Bacteria in ovarian tissue labeled with Nanogold and silver enhanced for 15 minutes to improve visualization by SEM. Scale bar = 0.5 μm.

    Article Snippet: Electron dense probes such as colloidal gold, Nanogold, Aurion Ultrasmall gold, or quantum dots, allow high spatial resolution of macromolecular structures to provide superior insights into cellular and subcellular composition and functional relationships.

    Techniques: Labeling

    Chlamydia infected HeLa cells were immune-labeled intracellularly for antigens against a bacterial surface protein using Nanogold followed by silver enhancement and viewed by either cryo-SEM (A) or TEM (B). Scale bar = 0.5 micron.

    Journal: Current Protocols in Microbiology

    Article Title: Scanning Electron Microscopy

    doi: 10.1002/9780471729259.mc02b02s25

    Figure Lengend Snippet: Chlamydia infected HeLa cells were immune-labeled intracellularly for antigens against a bacterial surface protein using Nanogold followed by silver enhancement and viewed by either cryo-SEM (A) or TEM (B). Scale bar = 0.5 micron.

    Article Snippet: Electron dense probes such as colloidal gold, Nanogold, Aurion Ultrasmall gold, or quantum dots, allow high spatial resolution of macromolecular structures to provide superior insights into cellular and subcellular composition and functional relationships.

    Techniques: Infection, Labeling, Transmission Electron Microscopy

    Fig. 6. Comparable ultrastructural localization of LYVE-1 in intestinal lymphatic vessels of wild-type and T1α/podoplanin –/– mice. ( A ) High levels of immuno-nanogold labeling for LYVE-1 were detected at both the luminal (L) and the abluminal plasma membrane of lymphatic endothelium in the ileum of newborn wild-type mice. Some labeling of the lateral plasma membranes was also observed, whereas LYVE-1 was absent from the cytoplasm. ( B ) LYVE-1 expression was absent from blood vascular endothelium. ( C and D ) Lymphatic endothelial cells in the intestine of T1 α /podoplanin –/– mice also had high levels of LYVE-1 immuno-nanogold labeling of the luminal and abluminal plasma membranes. The lateral plasma membranes were also labeled, with the exception of punctate contact areas between adjacent cells (A, C and D, arrows). ( E and F ) Replacement of the primary LYVE-1 antibody with an unrelated rabbit IgG control resulted in the absence of lymphatic endothelial cell labeling in the ileum of wild-type (E) and of T1 α /podoplanin -null mice (F). Bars for (A), (C), (D) and (E) = 0.5 µm, (B) and (F) = 0.4 µm.

    Journal: The EMBO Journal

    Article Title: T1?/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

    doi: 10.1093/emboj/cdg342

    Figure Lengend Snippet: Fig. 6. Comparable ultrastructural localization of LYVE-1 in intestinal lymphatic vessels of wild-type and T1α/podoplanin –/– mice. ( A ) High levels of immuno-nanogold labeling for LYVE-1 were detected at both the luminal (L) and the abluminal plasma membrane of lymphatic endothelium in the ileum of newborn wild-type mice. Some labeling of the lateral plasma membranes was also observed, whereas LYVE-1 was absent from the cytoplasm. ( B ) LYVE-1 expression was absent from blood vascular endothelium. ( C and D ) Lymphatic endothelial cells in the intestine of T1 α /podoplanin –/– mice also had high levels of LYVE-1 immuno-nanogold labeling of the luminal and abluminal plasma membranes. The lateral plasma membranes were also labeled, with the exception of punctate contact areas between adjacent cells (A, C and D, arrows). ( E and F ) Replacement of the primary LYVE-1 antibody with an unrelated rabbit IgG control resulted in the absence of lymphatic endothelial cell labeling in the ileum of wild-type (E) and of T1 α /podoplanin -null mice (F). Bars for (A), (C), (D) and (E) = 0.5 µm, (B) and (F) = 0.4 µm.

    Article Snippet: After washing, sections were immersed in normal goat serum (Vector Laboratories, Burlingame, CA) and incubated with anti-LYVE-1 or 8.1.1 antibody, followed by incubation with goat anti-rabbit Fab′, conjugated with 1.4 nm nanogold particles (Nanoprobes, Stony Brook, NY), or with goat anti-hamster IgG, conjugated with 0.8 nm nanogold particles (Aurion, Wageningen, The Netherlands).

    Techniques: Mouse Assay, Labeling, Expressing

    Fig. 5. Ultrastructural localization of T1α/podoplanin in murine intestinal lymphatic vessels, but not in blood vessels, by immuno-nanogold staining. ( A ) In newborn wild-type mice, membrane-bound T1α/podoplanin was detected at the luminal (L) side of the lymphatic endothelium in the intestine (ileum). Fewer immuno-nanogold particles were observed at the abluminal plasma membrane and no T1α/podoplanin was detected within lymphatic endothelial cells. ( B ) T1α/podoplanin expression was completely absent from blood vascular endothelial cells in the intestine of newborn wild-type mice. ( C ) Absence of specific labeling of wild-type lymphatic endothelium after omission of the primary anti-T1α/podoplanin antibody. ( D ) Lymphatic endothelial cells of a newborn T1 α /podoplanin –/– mouse do not react with the anti-T1α/podoplanin antibody. Bars for (A) = 0.4 µm, (B) = 0.2 µm, (C) = 0.3 µm, (D) = 0.5 µm.

    Journal: The EMBO Journal

    Article Title: T1?/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

    doi: 10.1093/emboj/cdg342

    Figure Lengend Snippet: Fig. 5. Ultrastructural localization of T1α/podoplanin in murine intestinal lymphatic vessels, but not in blood vessels, by immuno-nanogold staining. ( A ) In newborn wild-type mice, membrane-bound T1α/podoplanin was detected at the luminal (L) side of the lymphatic endothelium in the intestine (ileum). Fewer immuno-nanogold particles were observed at the abluminal plasma membrane and no T1α/podoplanin was detected within lymphatic endothelial cells. ( B ) T1α/podoplanin expression was completely absent from blood vascular endothelial cells in the intestine of newborn wild-type mice. ( C ) Absence of specific labeling of wild-type lymphatic endothelium after omission of the primary anti-T1α/podoplanin antibody. ( D ) Lymphatic endothelial cells of a newborn T1 α /podoplanin –/– mouse do not react with the anti-T1α/podoplanin antibody. Bars for (A) = 0.4 µm, (B) = 0.2 µm, (C) = 0.3 µm, (D) = 0.5 µm.

    Article Snippet: After washing, sections were immersed in normal goat serum (Vector Laboratories, Burlingame, CA) and incubated with anti-LYVE-1 or 8.1.1 antibody, followed by incubation with goat anti-rabbit Fab′, conjugated with 1.4 nm nanogold particles (Nanoprobes, Stony Brook, NY), or with goat anti-hamster IgG, conjugated with 0.8 nm nanogold particles (Aurion, Wageningen, The Netherlands).

    Techniques: Staining, Mouse Assay, Expressing, Labeling

    Immunoelectron microscopic localization of APPL1. HeLa cells were labeled with antibodies to APPL1, followed by a Nanogold-labeled conjugate. Gold particles were visualized by silver enhancement. (A) A low-magnification view with two APPL-positive structures circled in orange, one of which is shown at higher magnification in the inset. (B–F) A gallery of representative structures. Dense labeling is associated with small tubular profiles close to the PM or deeper inside the cell as well as with larger heterogeneous structures. Note that labeling of the PM or CCPs is very low. M, mitochondria. Bars: (A, C, and E) 500 nm; (A [inset], B, D, and F) 200 nm.

    Journal: The Journal of Cell Biology

    Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

    doi: 10.1083/jcb.201311117

    Figure Lengend Snippet: Immunoelectron microscopic localization of APPL1. HeLa cells were labeled with antibodies to APPL1, followed by a Nanogold-labeled conjugate. Gold particles were visualized by silver enhancement. (A) A low-magnification view with two APPL-positive structures circled in orange, one of which is shown at higher magnification in the inset. (B–F) A gallery of representative structures. Dense labeling is associated with small tubular profiles close to the PM or deeper inside the cell as well as with larger heterogeneous structures. Note that labeling of the PM or CCPs is very low. M, mitochondria. Bars: (A, C, and E) 500 nm; (A [inset], B, D, and F) 200 nm.

    Article Snippet: After washing (6 × 10 min with PBS) cells were incubated with 1.4 nm goat anti-rabbit Nanogold (Nanoprobes) for 2 h at RT.

    Techniques: Labeling

    The filamentous nature of RRs is evident by EM staining and ARL2 and IMPDH2 colocalize to RRs as seen by immunogold staining. HeLa cells were induced with 1 μM MPA for 2 h and processed for EM, as described under Materials and Methods . All scale bars = 200 nm. (A) Longitudinal sections of RRs. The RRs are indicated by an asterisk (*). (B) A zoomed-out image of the RR shown in the far-right image from A. (C) Transverse sections of RRs. Labeling scheme is the same as A. (D) Immuno-EM showing localization of IMPDH2 using IMPDH2 antibody coupled to nanogold particles. The asterisk marks the RR while the black particles indicate the IMPDH2 localization. (E) Same as D except using ARL2 antibody to show localization of ARL2. (F) The fraction of RRs that were associated with the ER or mitochondrial membranes were counted in randomly acquired EM images of the RRs and plotted. N = 45.

    Journal: Molecular Biology of the Cell

    Article Title: Compositional complexity of rods and rings

    doi: 10.1091/mbc.E18-05-0274

    Figure Lengend Snippet: The filamentous nature of RRs is evident by EM staining and ARL2 and IMPDH2 colocalize to RRs as seen by immunogold staining. HeLa cells were induced with 1 μM MPA for 2 h and processed for EM, as described under Materials and Methods . All scale bars = 200 nm. (A) Longitudinal sections of RRs. The RRs are indicated by an asterisk (*). (B) A zoomed-out image of the RR shown in the far-right image from A. (C) Transverse sections of RRs. Labeling scheme is the same as A. (D) Immuno-EM showing localization of IMPDH2 using IMPDH2 antibody coupled to nanogold particles. The asterisk marks the RR while the black particles indicate the IMPDH2 localization. (E) Same as D except using ARL2 antibody to show localization of ARL2. (F) The fraction of RRs that were associated with the ER or mitochondrial membranes were counted in randomly acquired EM images of the RRs and plotted. N = 45.

    Article Snippet: The gold particles were then enhanced by nanogold enhancer (Nanoprobes) according to the manufacturer’s instructions.

    Techniques: Staining, Labeling