Tissues were fixed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer and post-fixed in OsO4 (2%) in a 0.1 M cacodylate buffer. After washing in 0.1 M cacodylate buffer, the cells were dehydrated through an ethanol series. The material was then penetrated by an increasing series of Spurr resin diluted in acetone. Finally, the tissues were embedded in Spurr resin. Both the thick and ultrathin sections were sectioned with an Ultracut Leica UC6 ultramicrotome (Leica Microsystems, Germany) using a diamond knife. Thick sections (0.5 μm) were placed on the slides, stained with Toluidine Blue, and imaged with a Leica DMRA2 fluorescence microscope using a Q-Imaging Retiga EX camera and Openlab 3.1.7 software. Ultrathin sections (80 nm) were placed onto copper grids coated with Formvar film, stained with uranyl acetate and lead citrate solutions, and examined with a Zeiss Libra 120 transmission electron microscope (Karl Zeiss Group, Germany).
Ultracut uc6 ultramicrotome
Manufactured by Leica
Sourced in Germany, United States
The Ultracut UC6 is a high-performance ultramicrotome designed for ultrathin sectioning of samples for electron microscopy. It features a fully motorized design, precise specimen advancement, and a robust construction for reliable and accurate sectioning.
Automatically generated - may contain errors
Lab products found in correlation
Tecnai g2 spirit tem, by Thermo Fisher Scientific (4 mentions)
Ht7700, by Hitachi (3 mentions)
Jem 1010 electron microscope, by JEOL (3 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (2 mentions)
Uranyl acetate, by Electron Microscopy Sciences (2 mentions)
Tecnai g2 spirit transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Epon resin, by Electron Microscopy Sciences (2 mentions)
Dmra2, by Leica (1 mentions)
12 nm colloidal gold conjugated goat anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
410 transmission electron microscope, by Philips (1 mentions)
Eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
Tecnai spirit electron microscope, by Thermo Fisher Scientific (1 mentions)
Pelco biowave, by Ted Pella (1 mentions)
Jem 1011, by JEOL (1 mentions)
G2 spirit tem, by Thermo Fisher Scientific (1 mentions)
Em pact2 high pressure freezer, by Leica (1 mentions)
Tecnai g2 spirit 120kv, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
43 protocols using ultracut uc6 ultramicrotome
1
Ultrastructural Analysis of Biological Samples
2
Ultrastructural Localization of VASA and CYTB
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Testes were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After dehydrating the samples through increasing concentrations of ethanol, the pellets were embedded into LR White Resin. The blocks were sectioned with an Ultracut Leica UC6 ultramicrotome (Leica Microsystems, Germany) using a diamond knife. The thin sections were mounted on Formvar-coated nickel grids. The grids were floated on drops of 1% BSA/0.01% Tween 20 in (PBST) for 1 h, then incubated for 1 h with primary antibodies: anti-VASA antibody in rabbit and anti-CYTB antibody in chicken, diluted with PBS-0.05% Tween 20 (PBST) at 1:200 concentration. Next, samples were washed in PBST and incubated for 2 h at RT with a mixture of secondary antibodies diluted 1:50 in PBST. The secondary antibodies used were: 12-nm colloidal gold-conjugated goat anti-rabbit IgG antibody (111-205-144, Jackson) and 18-nm colloidal gold-conjugated goat anti-chicken IgG antibody Jackson) . For control sample preparation, primary antibodies were omitted and only secondary antibodies were used. The grids were washed three times in PBST, rinsed in distilled water, stained with uranyl acetate and lead citrate, and observed and photographed with a Philips 410 Transmission Electron Microscope (Philips Electronics, Eindhoven, The Netherlands).
3
Transmission Electron Microscopy Sample Preparation
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Cells were fixed with 2.5% glutaraldehyde in phosphate buffer and kept in the fixative during 1 h at room temperature. They were washed and postfixed with 1% osmium tetroxide in the same buffer containing 0.8% potassium ferricyanide at 4°C. The samples were dehydrated in acetone, infiltrated with Epon resin during 2 days, embedded in the same resin and polymerised at 60°C during 48 h. Ultrathin sections were obtained using a Leica Ultracut UC6 ultramicrotome (Leica Microsystems, Vienna) and mounted on Formvar-coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate. Sections were observed in a Tecnai Spirit electron microscope equipped with an Eagle CCD camera (FEI, Eindhoven, The Netherlands).
4
Transmission Electron Microscopy of Sperm
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TEM was performed according to Ngo et al.65 (link). Briefly, sperms were fixed in 2.5% glutaraldehyde with 0.1 M HEPES pH 7.5 overnight. Subsequent processing was done using a Pelco Biowave (Ted Pella) processing microwave. The samples were post-fixed in 1% osmium tetroxide prior to dehydration in increasing ethanol gradient and then in combination with increasing ratio of LR White Resin before embedding in pure LR White Resin and polymerised at 60 °C for 24 h. Ultrathin sections (60 nm) were made using an Ultracut UC6 ultramicrotome (Leica Microsystems) with a diamond knife then placed on copper grids. The grids were post-stained with 5% uranyl acetate in 50% ethanol for 2 min, followed by Reynolds lead citrate for 30 s at room temperature. Ultrathin sections were examined under a JEM-1011 (JEOL) or HT7700 (Hitachi) transmission electron microscope, both operated at 80 kV.
5
Organoid Ultrastructural Characterization
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Therminox coverslips (Ted Pella 26028) were sterilized with 70% ethanol and UV irradiation for 1 h prior to coating in the usual fashion (Matrigel for Y6 iPSCs; vitronectin for ACS1028 iPSCs; and fibronectin for iPSC-ECs). Cells were plated and grown as described. At the desired time intervals, the coverslips were removed and placed in warmed (0.1M sodium cacodylate, pH 7.4, 2% paraformaldehyde, and 2.5% glutaraldehyde) and fixed for 60 min at 37°. Cells were then post-fixed in 2% osmium tetraoxide in imidazole buffer 0.1M (pH 7.5) for 1 h, rinsed in distilled water, stained with 3% UA for 1 h, and again rinsed in distilled water. Samples were then dehydrated in ascending grades of acetone, acetone and resin, embedded in a mixture of an Embed 812 kit (EMS 14120), and cured for 48 h in a 60 °C oven.
Ultrathin sections (70 nm) of the organoid were produced using a Leica UltraCut UC6 ultramicrotome, collected on 200 mesh copper grids and post-stained with 3% UA and Reynolds lead citrate. Micrographs were collected using on an FEI Technai Spirit G2 TEM.
Ultrathin sections (70 nm) of the organoid were produced using a Leica UltraCut UC6 ultramicrotome, collected on 200 mesh copper grids and post-stained with 3% UA and Reynolds lead citrate. Micrographs were collected using on an FEI Technai Spirit G2 TEM.
6
Ultrastructural Analysis of Hydrogels
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Cornea A1 and one half of a RHCIII and a RHCIII-MPC hydrogel underwent standard processing for TEM, whereby the specimen was dehydrated through an ascending ethanol series before being embedded in Araldite resin and polymerised at 60 °C for 24 h. The other half of the RHCIII and RHCIII-MPC hydrogels were processed for TEM using a high pressure freezing technique specifically designed to avoid preparation artefacts that may occur during standard TEM processing [29] (link). These samples were trimmed to 1.2 mm and rapidly cryofixed in a Leica EMPACT2 high pressure freezer. Frozen specimens were freeze substituted at −80 °C in 2% Osmium/Acetone for 24 h, and embedded in Araldite resin at 20 °C. From each embedded/frozen specimen, ultrathin sections (∼110 nm thick) were cut with a diamond knife on a Leica Ultracut UC6 ultramicrotome. Sections were collected on peloform-coated slot grids, stained with saturated aqueous uranyl acetate for 30 min at room temperature, Lead citrate for 7 min, and examined at 80 kV in a JEOL 1010 transmission electron microscope (Japan) equipped with a bottom-mount 14-bit CCD camera (Orius SC1000 Gatan, Pleasanton, CA).
7
Ultrastructural Analysis of Intracellular Chlamydia
8
Ultrastructural Analysis of Sciatic Nerve
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Sciatic nerve samples were submerged in fixative (0.1 M sodium cacodylate buffer pH 7.3 containing 2% paraformaldehyde and 2.5% glutaraldehyde). Semithin section and electron microscopic analyses were performed at the Center for Advance Microscopy at Northwestern University Feinberg School of Medicine. Samples were post-fixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, rinsed with distilled water, en bloc stained with 3% uranyl acetate, rinsed with distilled water, dehydrated in ascending grades of ethanol, transitioned with propylene oxide and embedded in resin mixture of Embed 812 kit, cured in a 60 °C oven, and sectioned on a Leica Ultracut UC6 ultramicrotome. 1 um thick sections were collected and stained with Toluidine Blue O. 70 nm sections were collected on 200 mesh Cu grids; thin sections were stained with uranyl acetate and Reynolds lead citrate.
9
Ultrastructural Muscle Damage Assessment
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Muscle biopsies were taken from pre-defined locations at three time points in the experiment: 1) T = 0; before vascular pedicle transection, serving as a normal control sample, 2) T = 12; at 12 h of flap preservation and 3) T = 36; at 36 h of flap preservation. Biopsies measured 2x4 mm and were stored in 2% Glutaraldehyde buffered with 0.1M Sodium,-Cacodylate pH 7.4 and postfixed in 0.5% Potassiumhexacyanoferrat (II)-Trihydrate in 1% Osmium tetroxide in Paladebuffer. Samples were dehydrated in ethanol, propylenoxid and embedded in Epon. Semithin sections (0.7μm) and ultrathin sections (80 nm) were cut using the Leica ultracut UC6 ultramicrotome. The ultrathin sections were examined using a Jeol TEM1400 transmission electron microscope. The electron microscopy images were photographed with a Catan digital micrograph camera. Five areas per biopsy were scored by two pathologists that were blinded to the method of flap preservation. A simple scoring system was designed to equally evaluate the samples, since none was available in literature. The degree of damage to myofibrils, mitochondria and capillaries were scored from 0 to 2 in this scoring system. A total score of 0 resembled no damage to the observed structure, a score of 2 resembled severe destruction (Table 1 ). A mean score (0 to 2) was calculated as a measure for overall muscle damage.
10
Ultrastructural Analysis of Blastocyst Efferocytosis
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Six blastocysts were randomly selected for ultrastructural examinations of efferocytosis by TEM (Fig. 5 ). All embryos were fixed in 2.0% paraformaldehyde and 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.3) at 4 °C for 60 min. They were then washed in 0.1 M sodium cacodylate buffer twice for 15 min and post-fixed in 1% OsO4 in 0.1 M sodium cacodylate buffer for 1 h at room temperature. The blastocysts were then individually embedded in 2% agar. After dehydrating by passing them through an acetone series (30, 50, 70, 80, 90 and 100%), they were embedded in Poly/Bed resin (Polysciences Inc., Warrington, PA, USA) and serially sectioned (c80 nm) using an Ultracut UC 6 ultramicrotome (Leica, Wetzlar, Germany). These ultra-thin sections were collected on mesh nickel grids, contrasted with uranyl acetate and lead citrate, and examined using a JEOL CX100 transmission electron microscope (JEOL, Tokyo, Japan)61 (link).
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