For transmission electron microscopy (TEM), the larvae were fixed in a mixed solution of 1 % paraformaldehyde (Sigma-Aldrich, Buchs, Switzerland) and 2.5 % glutaraldehyde (Sigma-Aldrich, Buchs, Switzerland) in 0.1 M sodium phosphate buffer, pH 7.5 at 4 °C overnight. Afterwards, the samples were prepared for embedding into epoxy resin and for transmission electron microscopy according to standard procedures. Pospischil et al., 1990). Epoxy resin blocks were screened for larvae using semithin sections (1 µm), which were stained with toluidine blue (Sigma-Aldrich, Buchs, Switzerland) to visualize tissue. Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich, Buchs, Switzerland) and lead citrate (Merck Eurolab AG, Dietlikon, Switzerland) and investigated using a Philips CM10 transmission electron microscope (Philips Electron Optics, Eindhoven, The Netherlands). Images were processed with Imaris (Bitplane AG, Zurich, Switzerland) and assembled for publication using Adobe Photoshop.
Uranyl acetate dihydrate
Manufactured by Merck Group
Sourced in Switzerland, United States
Uranyl acetate dihydrate is a chemical compound commonly used in various laboratory applications. It serves as a staining agent in electron microscopy, allowing for enhanced contrast and visualization of biological samples. The compound has a characteristic yellow crystalline appearance.
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Lab products found in correlation
Toluidine blue, by Merck Group (5 mentions)
Copper grid, by Merck Group (3 mentions)
Glutaraldehyde solution, by Merck Group (3 mentions)
Cm10 transmission electron microscope, by Philips (2 mentions)
Osmium tetroxide solution, by Merck Group (2 mentions)
Lead 2 citrate tribasic trihydrate, by Merck Group (2 mentions)
Fluka epoxy embedding medium kit, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Sodium hydrogen phosphate, by Merck Group (2 mentions)
Sodium dihydrogen phosphate, by Merck Group (2 mentions)
Ethanol, by Thermo Fisher Scientific (2 mentions)
Sodium hydrate, by Merck Group (2 mentions)
Polyoxyethylene 5 nonylphenylether np 5, by Merck Group (2 mentions)
Cyclohexane, by Thermo Fisher Scientific (2 mentions)
Calcium chloride, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Imaris, by Oxford Instruments (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Lead citrate, by Merck Group (1 mentions)
Trichloromethane, by Merck Group (1 mentions)
11 protocols using uranyl acetate dihydrate
1
Transmission Electron Microscopy of Larvae
2
Histological and Ultrastructural Analysis of Gill Samples
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Screening of gill samples with standard histology was performed on samples fixed in 10% neutral buffered formalin, followed by dehydration in an ascending alcohol series ending in xylol and afterwards embedded in paraffin. Paraffin blocks were cut in 2–3 μm thin sections, mounted on glass slides and stained using a routine protocol for haematoxylin and eosin (HE) staining.
For electron microscopy, formalin-fixed gill tissues were post-fixed in a mixed solution of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C overnight. Samples were prepared for TEM by embedding into epoxy resin according to standard procedures (Seth-Smith et al., 2016 (link)). Epoxy resin blocks were screened for epitheliocystis lesions using semithin sections (1 μm) which were stained with toluidine blue (Sigma-Aldrich). Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich), and lead citrate (Merck Eurolab AG) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris 7.6.1 (Bitplane, Oxford Instruments) and assembled into panels for publication and annotated using Photoshop (Adobe).
For electron microscopy, formalin-fixed gill tissues were post-fixed in a mixed solution of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C overnight. Samples were prepared for TEM by embedding into epoxy resin according to standard procedures (Seth-Smith et al., 2016 (link)). Epoxy resin blocks were screened for epitheliocystis lesions using semithin sections (1 μm) which were stained with toluidine blue (Sigma-Aldrich). Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich), and lead citrate (Merck Eurolab AG) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris 7.6.1 (Bitplane, Oxford Instruments) and assembled into panels for publication and annotated using Photoshop (Adobe).
3
Ultrastructural Analysis of Fish Gill Epitheliocystis
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Fish larvae fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4 °C were embedded into epoxy resin and prepared for TEM according to standard procedures13 (link). Gill sections containing epitheliocystis lesions were selected from epoxy resin blocks using semithin sections (1 μm) stained with toluidine blue (Sigma–Aldrich). Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma–Aldrich) and lead citrate (Merck Eurolab AG) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris 7.6.1 (Bitplane, Oxford Instruments) and assembled for publication using Photoshop CS4 extended, version 11.0.2 or CS6 extended, version 13.0 × 32 (Adobe).
4
Electron Microscopy of Larval Tissue
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For electron microscopy, larvae were fixed in a mixed solution of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C overnight. Samples were prepared by embedding into epoxy resin and for TEM microscopy according to standard procedures. Epoxy resin blocks were screened for larval location using semithin sections (1 μm) which were stained with toluidine blue (Sigma-Aldrich) to visualize tissue. Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich) and lead citrate (Merck Eurolab AG) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris (Bitplane) and assembled for publication using Adobe Photoshop.
5
Uranium Detection in Aqueous Solutions
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Analytical grade reagents, uranyl acetate dihydrate (UO2(CH3COO)2.2H2O), zirconium tetrachloride (ZrCl4), trimesic acid (BTC), glacial acetic acid (CH3COOH), trichloromethane (CHCl3), acetone (C3H6O), hydrochloric acid (HCl), methanol (CH3OH), isonicotinic acid (C6H5NO2), acetonitrile (C2H3N), N,N′-dimethyl formamide (DMF) and Arsenazo (III) were purchased from Sigma-Aldrich (MO, USA) without further purification. A stock standard solution of uranium (1000 mg L− 1) was prepared by dissolving uranyl acetate dihydrate in deionized water and daily solutions were prepared by its proper dilution. 1000 mg L− 1 of Arsenazo (III) was prepared in deionized water as a stock solution and used as ligand after proper dilution.
6
Ultrastructural Analysis of Tumor Tissue
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Tumor tissues were cut immediately after dissection into pieces of approximately 1 mm3 and fixed using a 2.5% glutaraldehyde solution (Sigma-Aldrich) in 0.1 M phosphate-buffered saline (pH 6.9). The samples were washed in the same buffer and transferred to a 1% osmium tetroxide solution (Sigma-Aldrich) in 0.1 M phosphate-buffered saline (pH 6.9) for 1 hour, then washed in distilled water, dehydrated in ethanol gradients, and impregnated with epoxy embedding resin (Fluka Epoxy embedding medium kit; Sigma-Aldrich). The next day, the samples were embedded in the same resin and baked for 24 hours at 36°C, then transferred to a 60°C incubator and baked for a further 24 hours. The blocks were cut into ultrathin sections (50–80 nm) using an ultramicrotome (Ultratome III; LKB Products, Uppsala, Sweden) and transferred onto 200-mesh copper grids (Agar Scientific Ltd, Stansted, UK). Sections were contrasted using uranyl acetate dihydrate (Sigma-Aldrich) and lead citrate [lead (II) citrate tribasic trihydrate; Sigma-Aldrich], and examined by TEM.
7
Ultrastructural Analysis of Tumor Tissue
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Tumor tissues were cut immediately after resection into pieces of about 1.5 mm3 and fixed in a 2.5% glutaraldehyde solution (Sigma-Aldrich) in 0.1 M PBS (pH 7) overnight. The samples were washed in the PBS and transferred to a 1% osmium tetroxide solution (Sigma-Aldrich) in 0.1 M PBS (pH 7) for 1 h, then washed in distilled water, dehydrated in ethanol gradients, and impregnated with epoxy embedding resin (Fluka Epoxy Embedding Medium Kit; Sigma-Aldrich). After 24 h, the samples were embedded in the same resin and baked for 24 h at 36 °C, then transferred to a 60 °C incubator and baked for a further 24 h. The blocks were cut into ultrathin sections (50 nm) using an ultramicrotome (Ultratome III; LKB Products, Vienna, Austria) and transferred onto TEM grids (Formvar on 3 mm 200 Mesh Cu Grids, Agar Scientific, Stansted, UK). Sections were contrasted using uranyl acetate dihydrate (Sigma-Aldrich) and lead (II) citrate tribasic trihydrate (Sigma-Aldrich), and examined by TEM.
8
Peptide Synthesis and Characterization
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All amino acids used, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), dichloromethane (DCM) and dimethyl formamide (DMF) were bought from AGTC Bioproducts. NovaPEG Rink amide resin was purchased from Merck Novabiochem. Diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), 3,5-bis(trifluoromethyl) benzoic acid, thioflavin T, monobasic potassium phosphate, dibasic sodium phosphate, glutaraldehyde solution and uranyl acetate dihydrate were purchased from Sigma-Aldrich. HPLCgrade acetonitrile and N-methylpyrrolidone (NMP) and analytical grade purity 1,1,1,3,3,3,-hexafluoroisopropanol (HFIP), piperidine and triisopropylsilane (TIS) were bought from Fisher Scientific. The buffer used was a sterile filtered 10 mM phosphate buffer adjusted to pH 7.4 prepared by dilution from a stock solution of 0.5 M phosphate buffer pH 7.4 in ultrapure water (Synergy UV Ultrapure water system).
9
Ultrastructure Analysis of Epitheliocystis
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Gill branches fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C and originating from fish positive in histology for epitheliocystis and in PCR for Chlamydiales, were prepared for embedding into epoxy resin and for TEM according to standard procedures. Gill sections containing epitheliocystis lesions were selected from epoxy resin blocks using semithin sections (1 µm) stained with toluidine blue (Sigma-Aldrich, Missouri, USA). Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich, USA) and lead citrate (Merck Eurolab AG, Dietikon, Zurich, Switzerland) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris 7.7.1 (Bitplane, Zurich, Switzerland) and assembled for publication using Adobe Photoshop.
10
Characterization of Functionalized Nanoparticles
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