For pre-embedding immuno-electron microscopy, GLAST-mGC3 and GLAST-mGC3;IP3R2−/− mice (16–17 weeks old) were transcardially perfused with 4 % paraformaldehyde/0.1 % glutaraldehyde in 0.1 M phosphate buffer (PB) under deep pentobarbital anesthesia. Brains, still within the skull, were postfixed in the same fixative solution for 4 h at 4° C. After post-fixation, brains were isolated and stored in 0.1 M PB with 0.05 % sodium azide at 4° C until further processed. After blocking with 5 % normal donkey serum in PBS, coronal sections (60 μm in thickness) were incubated overnight with rabbit anti-EGFP IgG (Frontier Institute) and then with anti-rabbit IgG conjugated to 1.4 nm gold particles (Thermo Fisher Scientific). Following silver enhancement (HQ silver, Nanoprobes), sections were osmificated, dehydrated and embedded in Epon 812 resin. Ultrathin sections (70 nm in thickness) were prepared with an ultramicrotome (Leica Ultracut UCT) and stained with 2 % uranyl acetate and 1 % lead citrate. Electron micrographs were taken with an H-7600 electron microscope (Hitachi, Tokyo, Japan).
Hq silver
Manufactured by Nanoprobes
Sourced in United States, Japan
The HQ Silver is a high-quality laboratory equipment designed for precise measurement and analysis. It features advanced sensor technology to provide accurate and reliable data. The core function of the HQ Silver is to facilitate precise quantitative analysis in a wide range of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
H 7100, by Hitachi (5 mentions)
Anti rabbit nanogold, by Nanoprobes (3 mentions)
Goat anti rabbit igg, by Nanoprobes (3 mentions)
1 nm gold particles, by Nanoprobes (3 mentions)
1.4 nm anti rabbit nanogold, by Nanoprobes (3 mentions)
Nanogold fab goat anti rabbit igg h l, by Nanoprobes (2 mentions)
Ultrascan 4000 ccd camera, by Ametek (2 mentions)
First light digital camera controller, by Ametek (2 mentions)
Jem 1230 transmission electron microscope, by JEOL (2 mentions)
Jem 1230, by JEOL (2 mentions)
Goat anti rat igg, by Nanoprobes (2 mentions)
Primary rabbit anti cd63, by Santa Cruz Biotechnology (2 mentions)
Lr white resin, by Electron Microscopy Sciences (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
H 7600 electron microscope, by Hitachi (1 mentions)
Ultracut uct, by Leica (1 mentions)
Periodic acid, by Merck Group (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
32 protocols using hq silver
1
Pre-embedding Immuno-EM of GLAST Mice
2
Comprehensive Fixation and Staining Protocol
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Chemicals were purchased from EMSdiasum (Hatfield, PA, USA): 32% (v/v) paraformaldehyde (formaldehyde) aqueous solution (# 15714-S); glutaraldehyde (#16210), osmium tetroxide (OsO4; #19100), epoxy resins (#14120 Embed 812, an Epon substitute), UranyLess™ staining solution (#22409), uranyl acetate (UA; #22400; note: uranyl compounds are radioactive, thus, subject to specific safety regulations as to purchase, handling, disposal); from Sigma-Aldrich: periodic acid (PA; #10450-60-9), thiosemicarbazide (TCH; #79-19-6), silver proteinate (SP; #9008-42-8), lead citrate (#15326), methyl cellulose (#M7027), paraformaldehyde powder (#158127), phosphate buffer solution (0.1 M; #5244); or from other sources (i.e., gelatin, sucrose both from food store), HQ-Silver® (#2012, from Nanoprobes, Yaphank, NY, USA).
3
Immunodetection of VGLUT3 in Hippocampus
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VGLUT3 was detected with an electron microscope with a post-embedding immunogold method as previously described (Gras et al., 2002 (link), 2008 (link)). Sagittal brain sections of the hippocampus were incubated with guinea pig anti-VGLUT3 antiserum (1:1000). Immunolabeling was detected with goat anti-guinea pig IgGs conjugated to gold particles (1.4 nm in diameter; Nanoprobes, NY, USA, 1:100). After post-fixation (1% glutaraldehyde), the immunogold signal was enhanced with a silver enhancement kit (HQ silver, Nanoprobes; NY, USA). Ultrathin sections were examined with a transmission electron microscope (EM 912 OMEGA, ZEISS).
4
Immunohistochemical Labeling of M1 Receptor and KCNQ Channels
5
Pre-embedding Immunogold Labeling of BDNF
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Fifty micro meter vibratome sections from WT and Bsn-mutants were processed for pre-embedding immunogold labeling as previously described (Dieni et al., 2012 (link)). Briefly, sections were snap frozen to increase antigenicity, blocked in 3% M.O.M. and incubated in anti-BDNF (20 μg/ml) for three nights at 4°C. After thorough washing with 50 mM TBS, sections were treated overnight at 4°C with 1.4 nm gold-conjugated mouse IgG (1:100). Bound gold particles were enhanced using a silver intensification kit (HQ Silver; Nanoprobes) and sections were then fixed for 10 min in 1% GA solution. Sections underwent osmification (0.5% OsO4 and 6.86% sucrose in 0.1 M PB) for 40 min, were treated with 1% uranyl acetate in 70% ethanol (EtOH) for 35 min, and dehydrated in increasing grades of EtOH. After washing in propylene oxide, the tissue was embedded in Durcupan (Fluka). Ultrathin sections (60 nm) of CA3 were cut and mounted on formvar-coated copper grids.
6
Exosome Immunogold Labeling Protocol
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Exosomes were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer pH 7.4 overnight and 20 µl of suspended exosome preparation was applied to a carbon-formvar coated 200 mesh nickel grid (Electron Microscopy Sciences) and allowed to stand 30 min. Excess sample was wicked off onto Whatman filter paper. Grids were then floated exosome side down on 20 µl drops of 1 M ammonium chloride for 30 min to quench aldehyde groups from the fixation step, followed by flotation on drops of blocking buffer (Electron Microscopy Sciences) for 1 h. After three rinses in PBS, grids were incubated for 1 h on drops of 1.4 nm anti-rabbit nanogold (Nanoprobes, Inc.) diluted 1:1000 in blocking buffer, then washed in PBS three times, for 5 min each wash, followed by three washes in deionized H2O. Nanogold was enhanced for 8 min in HQ Silver, (Nanoprobes, Inc.) and rinsed in ice cold deionized H2O to terminate enhancement. Grids were then negatively stained in 2% aqueous uranyl acetate and allowed to air dry before being imaged in a JEM 1400Flash transmission electron microscope (JEOL USA Inc.) at 120 kV with a Gatan One View Digital Camera (Gatan Inc.).
7
Immunogold Labeling of Chromosomes
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Immunogold labelling was performed as described15 (link) with some modifications. PA chromosomes were dropped onto an aluminum foil substrates and treated with 0.5% triton X-100 in XBE2 (10 mM HEPES pH 7.7, 100 mM KCl, 5 mM EGTA, 2 mM MgCl2) for 10 min. Chromosomes were then blocked in 1% BSA in XBE2 for 30 min and incubated with Topo IIα antibody (1:50; Topogen) or hCAP-E (1:100) for 1 h. After washing, chromosomes were incubated with anti-mouse FluoroNanogold®or anti-rabbit FluoroNanogold® (1:200; Nanoprobes) for 1 h. The chromosomes were post-fixed with 2.5% glutaraldehyde in XBE5 (10 mM HEPES pH 7.7, 100 mM KCl, 5 mM EGTA, 5 mM MgCl2). After washing, samples were silver-enhanced (HQ silver, Nanoprobes). The immunogold-labelled chromosomes were then treated with 2% osmium tetroxide and extensively washed with ultrapure water. Sample dehydration was carried out using an ethanol series (70%, 100% and 100%) and then treatment with 3-methylbutyl acetate and critical point drying, after which osmium coating was performed.
8
Ultrastructural Analysis of Plant Root Tips
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For ultrastructural analysis, root tips were high-pressure frozen (Bal-Tec HPM010; Balzers) in hexadecene (Merck Sharp and Dohme, Haar, Germany), freeze-substituted in acetone containing 2.5% osmium tetroxide, washed at 0°C with acetone, and embedded in Epon. For immunogold labeling of ultrathin thawed cryosections, root tips were fixed with 8% formaldehyde (2 hr), embedded in gelatin, and infiltrated with 2.1 M sucrose in PBS as previously described (Dettmer et al., 2006 (link)). Thawed ultrathin sections were labeled with rabbit anti-GFP antibodies (1:300; Abcam) and silver-enhanced (HQ Silver, 8 min; Nanoprobes, Yaphank, NY, USA) goat anti-rabbit IgG coupled to Nanogold (no. 2004; Nanoprobes). Antibodies and markers were diluted in blocking buffer (PBS supplemented with 0.5% BSA and 1% milk powder).
9
Quantifying Chlamydia CPAF Localization
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HeLa cells were seeded in 24 well plates onto coverslips and infected with C. trachomatis L2 for 30 h. The cells were fixed in 4% PFA/0, 1% glutaraldehyde (EM grade, Roth) for 20 minutes and stained over night with the mouse anti-CPAF antibody (1∶50) in 50 mM TBS/2% normal goat serum, washed with 50 mM TBS and stained over night with a gold-coupled anti-mouse antibody (1∶100, Nanoprobes). After silver intensification (HQ silver, Nanoprobes) and counterstain with 0.1% osmium tetroxide (Roth), samples were embedded in Durcupan (Fluka), subjected to ultra-thin-sectioning and analyzed with a Zeiss Leo TEM. Quantification of immunogold labeling was performed using ITEM software (Olympus, Germany). Briefly, pictures of infected cells were randomly taken, the regions of interest (inclusions or cytoplasm) were outlined and the number of gold particles was counted within these regions. Non infected, stained cells of the same culture dishes were taken as negative control.
10
Immunolocalization of APPL1 in HeLa Cells
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HeLa cells in 3 cm dishes were fixed in PBS containing 1% PFA/0.1% glutaraldehyde and 0.01% digitonin for 15 min at RT and then with 1% PFA in PBS for further 15 min. They were then washed and incubated with 50 mM glycine in PBS for 5 min and then with blocking solution (1% BSA, 0.1% digitonin, and 50 mM glycine in PBS) before incubation with rabbit anti-APPL1 diluted 1:100 in 0.2% BSA, 0.1% fish skin gelatin for 60 min at RT. After washing (6 × 10 min with PBS) cells were incubated with 1.4 nm goat anti-rabbit Nanogold (Nanoprobes) for 2 h at RT. After fixation for 5 min with 1% glutaraldehyde in PBS, cells were washed sequentially with PBS and then distilled water (3 × 5 min) before silver enhancement for 3 min at RT in the dark according to the manufacturer’s instructions (Nanoprobes HQ Silver). Cells were further processed following standard procedures and flat embedded in epon resin. Sections were cut parallel to the culture substratum and stained with uranyl acetate and lead citrate before viewing.
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