The cells were fixed in a mixture of 1.6% paraformaldehyde and 2.5% glutaraldehyde prepared with a phosphate buffer (0.1 M, pH 7.4) for 1.5 h at room temperature as described by Szokoli et al. (2016) (link). Alternatively, 2.5% glutaraldehyde diluted with 0.1M cacodylate buffer (pH 7.4) was used as a fixative. The cells were washed in the same buffer containing 12.5% sucrose and postfixed in 1.6% OsO4 (1 h at 4°C). Then the cells were dehydrated in ethanol gradient followed by ethanol/acetone mixture (1:1), 100% acetone and embedded in Epoxy embedding medium (FlukaChemie AG, St. Gallen, Switzerland) according to the manufacturer’s protocol. The blocks were sectioned with a Leica EM UC6 Ultracut and ultrathin sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate. All samples were examined with a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) or JEM-2000 (JEOL Ltd., Tokyo, Japan) at 90 kV.
Ultracut em uc6
Manufactured by Leica camera
Sourced in Germany, Austria
The Ultracut EM UC6 is a high-performance ultramicrotome designed for the preparation of ultra-thin sections for electron microscopy. It features precise thickness control, intuitive operation, and a durable construction for reliable performance in the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Jem 1400 electron microscope, by JEOL (2 mentions)
Imprinted 50 μm relocation grids, by Ibidi (2 mentions)
Technai 12 transmission electron microscope, by Philips (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Jem 2000, by JEOL (1 mentions)
Jem 1400, by JEOL (1 mentions)
12 well plate, by Sarstedt (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Quanta 650, by Thermo Fisher Scientific (1 mentions)
H 7000, by Hitachi (1 mentions)
Stereo discovery v8, by Zeiss (1 mentions)
Lsm 780 nlo, by Zeiss (1 mentions)
12 protocols using ultracut em uc6
1
Cell Fixation and Embedding for TEM
2
Histological Analysis of Nerve Regeneration
3
Electron Microscopy of Ciliates
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Ciliates were processed for electron microscopy as described elsewhere [59 (link)]. Briefly, the cells were fixed in a mixture of 1.6% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2–7.4) for 1.5 h at room temperature, washed in the same buffer containing sucrose (12.5%) and postfixed in 1.6% OsO4 (1 h at 4°C). Then the cells were dehydrated in an ethanol gradient followed by ethanol/acetone (1:1), 100% acetone, and embedded in Epoxy embedding medium (Fluka Chemie AG, St. Gallen, Switzerland). The resin was polymerized according to the manufacturer’s protocol. The blocks were sectioned with a Leica EM UC6 Ultracut. Sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate.
Negative staining was performed by first washing and starving the cells overnight in distilled water to decrease the abundance of food and environmental bacteria. Single cells were then squashed with the micropipette and the remaining were transferred onto grids covered with the supporting film. Staining was performed using aqueous 1% uranyl acetate. All samples were examined with a JEOL JEM-1400 (JEOL, Ltd., Tokyo, Japan) electron microscope at 90 kV. The images were obtained with an inbuilt digital camera.
Negative staining was performed by first washing and starving the cells overnight in distilled water to decrease the abundance of food and environmental bacteria. Single cells were then squashed with the micropipette and the remaining were transferred onto grids covered with the supporting film. Staining was performed using aqueous 1% uranyl acetate. All samples were examined with a JEOL JEM-1400 (JEOL, Ltd., Tokyo, Japan) electron microscope at 90 kV. The images were obtained with an inbuilt digital camera.
4
Ultrastructural Analysis of Paramecia Symbionts
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Paramecia were fixed in phosphate buffer (0.1 M, pH 7.4) containing 1.6% paraformaldehyde and 2.5% glutaraldehyde for 1.5 h at room temperature as described by Szokoli and colleagues [35] (link). Then the cells were washed in the same buffer containing 12.5% sucrose and postfixed in 1.6% OsO4
(1 h at 4 • C). After that, the cells were dehydrated in ethanol gradient followed by ethanol/acetone mixture (1:1), 100% acetone and embedded in Epoxy embedding medium (FlukaChemie AG, St. Gallen, Switzerland) according to the manufacturer's protocol. The blocks were sectioned with a Leica EM UC6 Ultracut, and ultrathin sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate. All samples were examined with a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) at 90 kV.
Negative staining was performed to test for the presence of flagella on the symbiont surface. For this purpose, the host cells were washed several times with sterile lettuce medium, incubated in it for 24 h and then washed again before the sample preparation. Several cells were squashed, and a drop of the resulting suspension was transferred to a Formvar coated grid. The bacteria were allowed to precipitate for about 1 min, and then the grid was covered with 1% uranyl acetate in water for about 1 min. Then the liquid was removed with filter paper, and the grid was air-dried.
(1 h at 4 • C). After that, the cells were dehydrated in ethanol gradient followed by ethanol/acetone mixture (1:1), 100% acetone and embedded in Epoxy embedding medium (FlukaChemie AG, St. Gallen, Switzerland) according to the manufacturer's protocol. The blocks were sectioned with a Leica EM UC6 Ultracut, and ultrathin sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate. All samples were examined with a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) at 90 kV.
Negative staining was performed to test for the presence of flagella on the symbiont surface. For this purpose, the host cells were washed several times with sterile lettuce medium, incubated in it for 24 h and then washed again before the sample preparation. Several cells were squashed, and a drop of the resulting suspension was transferred to a Formvar coated grid. The bacteria were allowed to precipitate for about 1 min, and then the grid was covered with 1% uranyl acetate in water for about 1 min. Then the liquid was removed with filter paper, and the grid was air-dried.
5
Ultrastructural Analysis of Macrophage-NP Interactions
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The RAW 264.7
macrophages were seeded in 12 well plates (Sarstedt), 24 h before
NP exposure (200 000 cells/well), and exposed to NPs with a concentration
of 50 μg/mL for 2 or 24 h. After incubation, the cells were
washed three times with PBS, detached with TrypLe (Invitrogen), pelleted,
and fixed with 0.9% NaCl solution containing 2.5% glutaraldehyde.
The pellets were postfixed in 2% osmium tetroxide in 0.1 M phosphate
buffer, pH 7.4 at 4 °C for 2 h, dehydrated in ethanol followed
by acetone, and embedded in LX-112 (Ladd, Burlington, Vermont, USA).
Ultrathin sections (∼70 nm) were cut using a Leica Ultracut
EM UC6 (Leica, Wien, Austria). The sections were contrasted with uranyl
acetate followed by lead citrate and examined by SEM (Quanta 650)
with a STEM II detector using 30 kV acceleration voltage (Thermo Fisher).
macrophages were seeded in 12 well plates (Sarstedt), 24 h before
NP exposure (200 000 cells/well), and exposed to NPs with a concentration
of 50 μg/mL for 2 or 24 h. After incubation, the cells were
washed three times with PBS, detached with TrypLe (Invitrogen), pelleted,
and fixed with 0.9% NaCl solution containing 2.5% glutaraldehyde.
The pellets were postfixed in 2% osmium tetroxide in 0.1 M phosphate
buffer, pH 7.4 at 4 °C for 2 h, dehydrated in ethanol followed
by acetone, and embedded in LX-112 (Ladd, Burlington, Vermont, USA).
Ultrathin sections (∼70 nm) were cut using a Leica Ultracut
EM UC6 (Leica, Wien, Austria). The sections were contrasted with uranyl
acetate followed by lead citrate and examined by SEM (Quanta 650)
with a STEM II detector using 30 kV acceleration voltage (Thermo Fisher).
6
Tissue Sectioning and Staining Protocol
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When the resin was completely polymerized, very small regions of interest were dissected with a blade under the stereomicroscope (ZEISS, SteREO Lumar.V12) and mounted on small pyramids of resin with glue. They were left to air-dry overnight.
The mounted tissue was placed on the ultramicrotome (Leica Ultracut EM UC6, Vienna, Austria) and with a razor-sharp blade, they were given the shape of a trapezoid. The tissue was then aligned with a glass knife and semithin sections of 1-µm-thickness were cut. Sections were collected with non-magnetic tweezers and placed in small drops of ddH2O on gelatinized glass slides. Glass slides were shortly placed on the heater and when the water drop was completely dried, then the tissue was contra stained with toluidine blue and observed with the brightfield microscope. Cutting of thin sections: Thin sections (70 nm thickness) were cut with a diamond blade and collected in ddH2O and gathered on a nickel grid (diameter: 3.05 mm, G300-Ni, 300 lines/inch square mesh, No. 100, Science Services GmbH, Munich, Germany). The grids were put in a grid box and air-dried overnight.
The mounted tissue was placed on the ultramicrotome (Leica Ultracut EM UC6, Vienna, Austria) and with a razor-sharp blade, they were given the shape of a trapezoid. The tissue was then aligned with a glass knife and semithin sections of 1-µm-thickness were cut. Sections were collected with non-magnetic tweezers and placed in small drops of ddH2O on gelatinized glass slides. Glass slides were shortly placed on the heater and when the water drop was completely dried, then the tissue was contra stained with toluidine blue and observed with the brightfield microscope. Cutting of thin sections: Thin sections (70 nm thickness) were cut with a diamond blade and collected in ddH2O and gathered on a nickel grid (diameter: 3.05 mm, G300-Ni, 300 lines/inch square mesh, No. 100, Science Services GmbH, Munich, Germany). The grids were put in a grid box and air-dried overnight.
7
Ultrastructural Analysis of Oyster Hemocytes
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TEM analysis of oyster hemocytes was performed following a previous study.29 (link) Hemocyte suspensions (1 × 106 cells) were centrifuged at 500 × g for 8 min at 4 °C, and the supernatant was discarded. Samples were fixed in 3% glutaraldehyde solution for 1 day at 4 °C. After 3 washes with 0.4 M cacodylate buffer, the cells were post-fixed with a solution of 1% osmium tetroxide for 1 h at 4 °C. Then, the cells were washed twice in 0.4 M cacodylate buffer. After dehydration in successive baths of ethanol and two baths of propylene oxide, samples were progressively impregnated and embedded in Epon. After polymerization at 60 °C, semi-thin sections were cut to 1 mm thickness for quality control and then to 80–85 nm for examination on a Leica Ultracut (EM UC6), floated onto copper EM grids, and stained with uracil acetate/Fahmys lead citrate. The sections were examined using a transmission electron microscope (H-7000, Hitachi, Japan).
8
Morphological Observations of Young Tree Leaves
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Morphological observations concerned the scale of the development of the above-ground part of the young trees, including the size and colour of leaves. The observations were conducted using a stereomicroscope (Zeiss SteREO Discovery.V8; Oberkochen, Germany). We focused on alterations in the colour and size of the leaves.
For microscopical observations, we always sampled the third leaf from the top of the stem of the trees growing both on mining sludge and in the control group. Next, small 3–5 mm long samples of leaves, together with the main vein near the top of the leaf, were cut off by a razor blade and rapidly placed in a glass bottle with a fixative solution (see below). Semi-thin sections (2.5 µm thickness) were cut using a diamond knife on an ultramicrotome (Leica Ultracut EM UC6, Vienna, Austria). Semi-thin sections were collected on basic glasses then dried (80 °C) and analysed using a confocal scanning laser microscope (LSM 510; Zeiss, Germany) in transmitted light.
For microscopical observations, we always sampled the third leaf from the top of the stem of the trees growing both on mining sludge and in the control group. Next, small 3–5 mm long samples of leaves, together with the main vein near the top of the leaf, were cut off by a razor blade and rapidly placed in a glass bottle with a fixative solution (see below). Semi-thin sections (2.5 µm thickness) were cut using a diamond knife on an ultramicrotome (Leica Ultracut EM UC6, Vienna, Austria). Semi-thin sections were collected on basic glasses then dried (80 °C) and analysed using a confocal scanning laser microscope (LSM 510; Zeiss, Germany) in transmitted light.
9
Correlative Light-Electron Microscopy of Ependymal Progenitors
10
Correlative Light-Electron Microscopy of Ependymal Progenitors
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CLEM was done as previously described12 (link). Briefly, primary CENT2-GFP ependymal progenitors were grown in 0.17-mm thick glass dishes with imprinted 50 μm relocation grids (Ibidi). At differentiation day 3–6, cells were fixed with 4% PFA for 10 min and ependymal progenitors undergoing A-phase or G-Phase were imaged for CENT2-GFP and DAPI, in PBS, with an upright epifluorescence microscope (Zeiss Axio Observer.Z1). Coordinates on the relocation grid of the cells of interest were recorded. Cells were then processed for transmission electron microscopy. Briefly, cultured cells were treated with 1% OsO4, washed and progressively dehydrated. The samples were then incubated in 1% uranyl acetate in 70% methanol, before final dehydration, pre-impregnation with ethanol/epon (2/1, 1/1, 1/2) and impregnation with epon resin. After mounting in epon blocks for 48 h at 60 °C to ensure polymerization, resin blocks were detached from the glass dish by several baths in liquid nitrogen. Using the grid pattern imprinted in the resin, 50 serial ultra-thin 70-nm sections of the squares of interest were cut on an ultramicrotome (Ultracut EM UC6, Leica) and transferred onto formvar-coated EM grids (0.4 × 2 mm slot). The central position of the square of interest and DAPI staining were used to relocate and image the cell of interest using a Philips Technai 12 transmission electron microscope.
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