Sample preparation for electron microscopy was described previously [7 (link)]. Briefly, deeply anesthetized mice were fixed in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4), and hippocampal slices (thickness, 100 μm) were sectioned. After washing with PB, slices were further fixed with 2% osmium tetroxide, stained with 2–4% uranyl acetate, and embedded in epoxy resin (Durcupan ACM-Fluka, Sigma). Ultra-thin sections (thickness, 70 nm) were counter stained with uranyl acetate and lead citrate, and images were captured with an electron microscope (H-7500, Hitachi). Images were analyzed with Image-J according to previously described parameters [12 (link)].
Durcupan acm fluka
Manufactured by Merck Group
Sourced in United States, Japan
Durcupan ACM Fluka is a laboratory resin product manufactured by Merck Group. It is designed for the embedding and preparation of biological samples for microscopy analysis. The product provides a medium for the embedding and stabilization of specimens, enabling their sectioning and examination under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
H 7500, by Hitachi (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Axiocam hrc digital camera, by Zeiss (1 mentions)
H 7500 transmission electron microscope, by Hitachi (1 mentions)
Cm10 transmission electron microscope, by Philips (1 mentions)
Reichert ultracut e, by Leica (1 mentions)
Orius sc600, by Ametek (1 mentions)
Biotinylated goat anti mouse igg antibody, by Vector Laboratories (1 mentions)
Avidin biotin peroxidase complex abc kit, by Vector Laboratories (1 mentions)
Goat anti rabbit fab fragment, by Nanoprobes (1 mentions)
Reelin, by Merck Group (1 mentions)
Hq silver kit, by Nanoprobes (1 mentions)
1.4 nm gold, by Nanoprobes (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Jem 1230 transmission electron microscope, by JEOL (1 mentions)
Ab6719, by Abcam (1 mentions)
Dp70 digital camera, by Olympus (1 mentions)
Fv300 confocal microscope, by Olympus (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
11 protocols using durcupan acm fluka
1
Ultrastructural Analysis of Hippocampal Tissue
2
Transmission Electron Microscopy Vascular Cell Analysis
3
Ultrastructural Examination of Ischemic Muscle
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After the desired time of ischemia or ischemia and reperfusion the right extremities were perfused through an intra-arterial catheter with warm (37°C) 4% paraformaldehyde in 0.1 M phosphate buffer (PB), followed by cold 2% glutaraldehyde solution (2% GA in 0.1 M PB) for a total of 30 minutes. Approximately 1×1 mm pieces of muscles were cut out and post-fixed in 2% glutaraldehyde (1 hour) followed by 1% osmium-tetroxide (in 0.1 M cacodylate buffer, 1 hour at 4°C). All pieces were dehydrated in graded alcohol series and embedded in araldite (Durcupan ACM Fluka, Sigma-Aldrich, St. Louis, MO). Ultrathin sections were prepared with an ultramicrotome, contrast-stained with uranyl acetate and lead citrate, and analyzed using a Hitachi H7500 transmission electron microscope (Hitachi Ltd, Tokyo, Japan). Electron micrographs were taken by an Olympus-SIS digital camera (Megaview II).
Semithin sections stained with toluidine blue were also prepared for light microscopic examination and inspected with a Zeiss Axiophot microscope equipped with AxioCamHRc digital camera (Carl Zeiss, Oberkochen, Germany).
The final montages from the pictures were prepared using Adobe Photoshop 7.0 (San Diego, CA) program.
Semithin sections stained with toluidine blue were also prepared for light microscopic examination and inspected with a Zeiss Axiophot microscope equipped with AxioCamHRc digital camera (Carl Zeiss, Oberkochen, Germany).
The final montages from the pictures were prepared using Adobe Photoshop 7.0 (San Diego, CA) program.
4
Ultrastructural Analysis of Biological Specimens
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One specimen was immediately fixed in 2.5% glutaraldehyde 0.2 M in Sørensen's phosphate buffer (pH 7.2) at +4°C for 4 h, washed with 0.2 M Sørensen's phosphate buffer (pH 7.2), and postfixed in 1% osmium tetroxide (OsO4) in 0.2 M Sørensen's phosphate buffer (pH 7.2) at +4°C for 1 h. The specimen was dehydrated in graded ethanol and acetone and flatembedded in embedding agent (Durcupan ACM Fluka, Sigma-Aldrich, St. Louis, MO, USA) for a better orientation. Semithin sections (1 μm) were cut with a LKB Ultrotome V ultramicrotome, stained with an aqueous solution of 1% toluidine blue in 1% borax and 1% pironine, and viewed and photographed with a Zeiss Primo Star light microscope (LM). From the same specimens used for LM, ultrathin sections of gold-silver interference color were cut with a diamond knife on a LKB Ultrotome V ultramicrotome, collected on uncoated 200–300 mesh copper grids, and stained with methanolic uranyl acetate and lead citrate. Micrographs were taken with a Philips CM-10 transmission electron microscope (TEM) at 80 kV.
5
Double Immunoelectron Microscopy of GFAP and Reelin
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For double immunoelectron microscopy, sections were cryoprotected and freeze-thawed. After blocking in 20% NGS in 50 mM TBS, sections were incubated with primary antibodies for GFAP (rabbit, polyclonal, DAKO) and Reelin (mouse-monoclonal, clone G10, Millipore, Billerica, MA, USA) in 50 mM TBS containing 3% NGS (Vector Laboratories, Burlingame, CA) for 24h at 4°C. After washes in TBS, the sections were incubated with biotinylated goat anti-mouse IgG antibody (1:100; Vector Laboratories) and goat anti-rabbit (Fab fragment, 1:100) coupled to 1.4 nm gold (Nanoprobes, Stony Brook, NY). Subsequently, sections were processed for silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes) and incubated with avidin-biotin peroxidase complex (ABC kit; Vector Laboratories) that was visualized with 3,3′-diaminobenzidine tetrahydrochloride (0.05%) as a chromogen and 0.01% H2O2 as substrate. Sections were then treated with 1% osmium tetroxide and uranyl acetate, dehydrated and flat-embedded in epoxy resin (Durcupan ACM Fluka; Sigma-Aldrich). Ultrathin sections were cut at 60–70 nm on an ultramicrotome (Reichert Ultracut E; Leica), and viewed on a Philips CM100 electron microscope. Images were taken with a CCD camera (Orius SC600; GATAN) and analyzed using GATAN imaging software.
6
Tetrahymena Cell Ultrastructure Analysis
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Tetrahymena cells were grown to a mid log phase. A 5-ml amount of cells was collected, washed with 10 mM Tris-HCl, pH 7.5, and fixed with 2% glutaraldehyde (Sigma-Aldrich; diluted from 8% with 0.1 M cacodylate buffer, pH 7.2) on ice with gentle rocking. After 1 h, 10 μl of freshly prepared 1% tannic acid was added, and samples were incubated for an additional 1 h. After five washes for 10 min each wash in cold 0.1 M cacodylate buffer, pH 7.2, samples were postfixed on ice for 1 h with 1% osmium tetroxide (Sigma-Aldrich) in ultrapure water. After five washes for 10 min each wash with water at room temperature, samples were embedded in 2% low–melting point agarose, and chunks were cut into 2- to 3-mm fragments and dehydrated in a percentage series of ethanol followed by infiltration in Durcupan ACM Fluka (Sigma-Aldrich) according to the manufacturer's instructions.
To study isolated cilia, 150 ml of wild-type or knockout culture was grown to a density of 3 × 105 cells/ml and deciliated with a pH shock, and cilia were recovered (Wloga et al., 2008 (link)). Cilia were fixed as described and sectioned (40- to 50-nm-thick sections) as described (Angus et al., 2001 (link)). All samples were analyzed using a JEM 1200 EX transmission electron microscope (JOEL, Japan).
To study isolated cilia, 150 ml of wild-type or knockout culture was grown to a density of 3 × 105 cells/ml and deciliated with a pH shock, and cilia were recovered (Wloga et al., 2008 (link)). Cilia were fixed as described and sectioned (40- to 50-nm-thick sections) as described (Angus et al., 2001 (link)). All samples were analyzed using a JEM 1200 EX transmission electron microscope (JOEL, Japan).
7
Ultrastructural GLT1 localization in SN
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Free-floating vibratome sections were successively incubated 1h at room temperature in 10% normal donkey serum in PBS and overnight at 4 °C with the anti-GLT1 antibody (1/1000 in PBS).
After extensive washes in PBS, sections were incubated 1 h at room temperature with a donkey anti-guinea pig biotinylated secondary antibody (1/200 in PBS, Jackson ImmunoResearch Europe, Cambridge, UK) followed by avidin-biotin-peroxidase complex (ABC kit, Vector Laboratory, Burlingame, CA, USA, 1 h at room temperature). Peroxidase activity was revealed using 3,3diaminobenzidine tetrahydrochloride (0,05% in PB). Sections were then stained with 1% osmium tetroxide in PB (45 min) followed by 0.5% uranyl acetate in distilled water (45 min), dehydrated in increasing alcohol concentrations then propylene oxide and flat embedded in Durcupan ACM Fluka (Sigma-Aldrich). After polymerization of the resin, the SN was dissected and re-embedded in Durcupan blocks. Ultrathin sections (80 nm) were obtained on a Reichert Ultracut ultramicrotome and collected on formvar-coated single slot grids. Because of the limited penetration of the GLT1 antibody (no more than 3-4 µm from the surface of the tissue), ultrathin sections were taken from the most superficial portions of immunoperoxidase-stained tissue.
After extensive washes in PBS, sections were incubated 1 h at room temperature with a donkey anti-guinea pig biotinylated secondary antibody (1/200 in PBS, Jackson ImmunoResearch Europe, Cambridge, UK) followed by avidin-biotin-peroxidase complex (ABC kit, Vector Laboratory, Burlingame, CA, USA, 1 h at room temperature). Peroxidase activity was revealed using 3,3diaminobenzidine tetrahydrochloride (0,05% in PB). Sections were then stained with 1% osmium tetroxide in PB (45 min) followed by 0.5% uranyl acetate in distilled water (45 min), dehydrated in increasing alcohol concentrations then propylene oxide and flat embedded in Durcupan ACM Fluka (Sigma-Aldrich). After polymerization of the resin, the SN was dissected and re-embedded in Durcupan blocks. Ultrathin sections (80 nm) were obtained on a Reichert Ultracut ultramicrotome and collected on formvar-coated single slot grids. Because of the limited penetration of the GLT1 antibody (no more than 3-4 µm from the surface of the tissue), ultrathin sections were taken from the most superficial portions of immunoperoxidase-stained tissue.
8
Ultrastructural Analysis of Spinal Cord Injury
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Mice were sacrificed and perfused transcardially with 4% paraformaldehyde and 0.5% glutaraldehyde in a 0.1 mol phosphate buffer,24 h post-SCIRI. The spinal cord tissue (T8-L4) was collected and coronal sections (100 μm) were cut by a vibratome, and postfixed with 4% glutaraldehyde in a 0.1 mmol cacodylate buffer (pH 7.4) for 1 h and incubated with 1% osmium tetroxide in a 0.1 mmol cacodylate buffer for 2 h. Spinal cord sections were dehydrated by an ascending series of ethanol and dry acetone and then embedded in Durcupan ACM Fluka (Sigma-Aldrich).Ultrathin sections (0.1 μm) were stained with uranyl acetate and lead citrate and subsequently examined with a JEOL JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan).
9
Ultrastructural Analysis of Murine Aorta
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Male mice were euthanized and transcardially perfused with saline followed by fixation solution (50 ml 4% PFA in PBS). Dissected aortas were additionally fixed in 3% glutaraldehyde for 24hs and embedded in resin (Durcupan ACM Fluka, Sigma-Aldrich) for transverse sectioning. Ultrathin sections were cut and counterstained at the Electron Microscopy facility (SIdI service) at the Universidad Autónoma de Madrid. Sections were imaged with a transmission electron microscope (Jeol Jem 1010, 80 kV, Jeol Ltd. Tokyo, Japan) and recorded with a Gatan camera (Orius, SC200W, Pleasenton, California) at the indicated magnifications.
10
Optimizing Leptotene-Zygotene Enrichment in Testis
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In order to determine the optimal age for LZ enrichment in the absence of PS contamination, whole testes of pups ranging from 9 to 14 dpp (sampled at one-day age differences) were fixed in 0.1 M phosphate buffer (pH 6.8) containing 2.5 % glutaraldehyde, post-fixed in 1 % osmium tetroxide, and embedded in Epon (Durcupan ACM Fluka, Sigma-Aldrich, St. Louis, MO) as previously reported [19 (link)]. 1 μm sections were cut with a Power Tome XL Ultra-microtome (Boeckeler Instruments, Tucson, AZ), stained with toluidine blue, and examined by light microscopy with an Olympus FV300 confocal microscope. Photographs were taken with an Olympus DP70 digital camera using the DPController v. 1.1.1.65 software.
To confirm the purity of each meiotic prophase sorted fraction, an aliquot of each classified fraction of 4C cells (LZ and PS) was immunolabeled with a rabbit antibody raised against the C-terminal region of mouse SYCP3 (Acris Antibodies GmbH, Herford, Germany; RA25051, 1:100) and Texas Red-tagged goat anti-rabbit secondary antibody (Abcam, Cambridge, MA; ab6719, 1:500) as described earlier [19 (link)]. Fluorescent images were acquired with the Fluoview v.4.3 software.
To confirm the purity of each meiotic prophase sorted fraction, an aliquot of each classified fraction of 4C cells (LZ and PS) was immunolabeled with a rabbit antibody raised against the C-terminal region of mouse SYCP3 (Acris Antibodies GmbH, Herford, Germany; RA25051, 1:100) and Texas Red-tagged goat anti-rabbit secondary antibody (Abcam, Cambridge, MA; ab6719, 1:500) as described earlier [19 (link)]. Fluorescent images were acquired with the Fluoview v.4.3 software.
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