For electron microscopy, after development of the immunoperoxidase reaction, sections were first treated with 1% OsO4 in 0.1 M PB for 10 min in the dark, on ice, and then dehydrated in an ascending series of ethanol solutions, followed by acetonitrile. An additional treatment with uranyl acetate (1% in 70% ethanol for 10 min in the dark, on ice) was included during the dehydration process. Sections were embedded in Durcupan (ACM, Fluka, Buchs, Switzerland). Areas of interest containing the dorsolateral fasciculus (Lissauer’s tract) and the superficial laminae were cut from the dorsal horn of lumbar segments of both MGL+/+ and MGL−/− spinal cords, and re-sectioned to produce ultrathin 50 nm thin sections with a Leica EM UC6 Ultramicrotome (Leica Microsystems). These sections were collected on a Formvar coated single-slot copper grid, contrasted with lead citrate (Ultrostain2, Leica), and examined with a Hitachi 7100 electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan). Electron micrographs at 40,000× magnification were acquired with a Veleta CCD camera (Olympus Soft Imaging Solutions, Germany).
Ultrostain 2
Manufactured by Leica
Sourced in Japan
The Ultrostain 2 is a compact and versatile tissue staining system designed for use in histology and pathology laboratories. It provides automated staining of paraffin-embedded tissue sections with a range of staining protocols. The Ultrostain 2 ensures consistent and reliable staining results, enabling efficient and standardized sample preparation for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Em uc6 ultramicrotome, by Leica (2 mentions)
7100 electron microscope, by Hitachi (2 mentions)
Veleta ccd camera, by Olympus (2 mentions)
Diatome diamond knife, by Electron Microscopy Sciences (1 mentions)
Uc6 ultramicrotome, by Electron Microscopy Sciences (1 mentions)
Ultracut uct, by Leica (1 mentions)
Em 208 electron microscope, by Philips (1 mentions)
Ultracut uct microtome, by Leica (1 mentions)
Em ac20, by Leica (1 mentions)
6 protocols using ultrostain 2
1
Ultrastructural Analysis of Spinal Cord
2
Ultrastructural Analysis of Dorsal Horn
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For electron microscopy, after development of the immunoperoxidase reaction, sections were first treated with 1% OsO4 in 0.1 m PB for 10 min in the dark, on ice, and then dehydrated in an ascending series of ethanol solutions, followed by acetonitrile. An additional treatment with uranyl acetate (1% in 70% ethanol for 10 min in the dark, on ice) was included during the dehydration process. Sections were embedded in Durcupan (ACM, Fluka, Buchs, Switzerland). Areas of interest containing the dorsolateral fasciculus (Lissauer’s tract) and the superficial laminae were cut from the dorsal horn of lumbar segments of both MGL+/+ and MGL−/− spinal cords, and re-sectioned to produce ultrathin 50-nm thin sections with a Leica EM UC6 Ultramicrotome (Leica Microsystems). These sections were collected on a Formvar-coated single-slot copper grid, contrasted with lead citrate (Ultrostain2; Leica), and examined with a Hitachi 7100 electron microscope (Hitachi High-Technologies, Tokyo, Japan). Electron micrographs at 40 000 × magnification were acquired with a Veleta CCD camera (Olympus Soft Imaging Solutions, Munster, Germany).
3
Ultrastructural Analysis of Biological Samples
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Samples were pinned out on Sylgard blocks, cut open longitudinally, and pinned out in an en face preparation. Samples were then transferred to 4% paraformaldehyde/2.5% glutaraldehyde in 0.1M phosphate buffer (72 mM Na2 HPO4 0.2 H2O, 28 mM NaH2PO4 0.2 H2O in distilled water [pH 7.2]) for 4 h at room temperature and then stored in 1:10 solution at 4 °C prior to processing. Processing was carried out in the Pelco Biowave Pro+. Samples were washed in 100 mM cacodylate buffer followed by secondary fixation and heavy metal staining with 1% OsO4 and 0.5% K3Fe(CN)6. Samples were then washed with dH2O followed by dehydration with ethanol (50, 70, 95%) and then acetone prior to gradual infiltration of Spurr’s resin (TAAB) (10, 30, 50, 70, 90% in acetone). After infiltration was completed, samples were cut from Sylgard blocks and embedded. The resin was polymerized in a 60 °C oven for 24 h. Next, 250-nm sections were cut using a Leica UC6 ultramicrotome and Diatome diamond knife, and sections were collected on 100 square mesh grids and counterstained with Uranyless (TAAB) and Ultrostain 2 (Leica) using a Leica AC20. Tomograms were acquired on a Jeol 1400+ with an AMT UltraVUE camera.
4
Ultrastructural Analysis of Muscle and C. elegans
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Muscle tissue from the proband, a human muscle control sample, C.elegansvab-10 RNAi and control worms were fixated in 2% glutaraldehyde/PBS, stained with 1% osmiumtetraoxide/PBS, dehydrated and embedded in epon (epoxyresin). Ultrathin sections (60 nm) were cut on a Leica Ultracut UCT and placed on a grid, contrasted using 3% uranylacetate/H2O and leadcitrate (Leica Ultrostain 2) and analyzed with transmission electron microscopy on a Philips EM 208 electron microscope.
5
Ultrastructural Analysis of A549 Cells
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A549 cells were treated either with vehicle (DMSO) or KHS101 10 μM for 48 and 96 h. After treatment the cells were fixed with 2% (v/v) glutaraldehyde in 0.04 M phosphate buffer pH 7.4 for 60 min, scraped off, collected in a tube and post-fixed with the same fixative for 24 h. The cells were then washed in PBS and resuspended in 15% BSA for 10 min. The cells were centrifuged, and the cell pellet fixed by slowly adding the fixative for 48 h at 4 °C. The cell pellets were cut in smaller pieces and prepared for EM like following. Pellet fragments were washed in phosphate buffer, stained with 1% osmium tetraoxide in phosphate buffer for 90 min, dehydrated in ethanol series and acetone and embedded in TAAB 812 Embedding Resin (T030, TAAB). Semithin (1 µm) sections were cut on Leica Ultracut UCT microtome and stained with toluidine blue. Ultrathin (70 nm) sections were collected on uncoated nickel grids (M200-NI, Electron Microscope Sciences). The grids were stained with 3% uranyl acetate for 15 min at 60 °C and 3% lead citrate (Leica Ultrostain 2) for 6 min at room temperature. The sections were analyzed with JEM-1400 Plus electron microscope, equipped with Quemsa TEM CCD camera and images obtained using Radius software (TEM Imaging Platform software).
6
Transmission Electron Microscopy Sample Preparation
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Epon-embedded samples were first cut into semithin 0.5 µm-thick sections. Sections were stained with toluidine and safranin O solution and, then, visualised using the light microscope to localise the ROI for further electron microscope investigation. Then, ultra-thin sections in the range of 60-80 nm thickness were cut from the localised region using an ultra-microtome (Reichert-Jung ultra-cut 701701, Leica microsystems, Wetzlar, Germany) equipped with a diamond knife (DU3525, Diatome, Nidau, Switzerland). Subsequently, sections were contrasted with 0.5 % uranyl acetate (Ultrostain 1, Laurylab, Brindas, France) for 30 min and 3 % lead citrate (Ultrostain 2, Leica) for 80 s using the Leica contrasting machine (Leica EM AC20) according to the manufacturer's protocol.
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