To observe ultrastructure of the small intestinal enterocytes, ultrathin sections of the duodenum were prepared with an ultramicrotome (Leica Biosystems). Briefly, the harvested samples were fixed with 2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed with 2% osmium tetroxide after several washes with PBS. After dehydration with increasing concentrations of ethanol and infiltration with propylene oxide, resin embedding was performed. The ultrathin sections were then double-stained with uranyl acetate, and lead citrate and images were obtained using a transmission electron microscope (Hitachi, Ltd., Tokyo, Japan) with 80 kV accelerating voltage.
Ultramicrotome
Manufactured by Leica Biosystems
Sourced in Germany
The Ultramicrotome is a precision instrument used in the preparation of ultrathin sections for electron microscopy. It precisely cuts thin slices of biological samples, enabling the examination of their internal structures at the cellular and subcellular levels.
Automatically generated - may contain errors
Lab products found in correlation
Lead stain solution, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (2 mentions)
Transmission electron microscope, by Hitachi (1 mentions)
Jem 1400, by JEOL (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Embed 812, by Electron Microscopy Sciences (1 mentions)
Jem 1010 transmission electron microscope, by JEOL (1 mentions)
Paraffin wax, by Carl Roth (1 mentions)
Xc50 camera, by Olympus (1 mentions)
Transmission electron microscope, by JEOL (1 mentions)
Jem 1400 plus transmission electron microscope, by JEOL (1 mentions)
Rabbit anti gfp antibody, by Abcam (1 mentions)
Spurr resin, by Electron Microscopy Sciences (1 mentions)
Leica tcs sp8 laser confocal microscope, by Leica Microsystems (1 mentions)
9 protocols using ultramicrotome
1
Ultrastructural Analysis of Intestinal Enterocytes
2
Tissue Fixation and Embedding for TEM Analysis
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The tissue was fixed in a fixative containing 2.5% glutaraldehyde and 3 mM CaCl3 in a 0.1 M cacodylate buffer for 1 h at room temperature. After a washing with a 0.1 M cacodylate buffer containing 3 mM CaCl3, the tissue was post-fixed for 40 min at 4 °C in a 0.1 M cacodylate buffer containing 1% osmium tetroxide and 1.5% potassium ferricyanide. Samples were washed briefly in distilled water, followed by gradual dehydration in a graded ethanol series of 70%, 90%, 95% (15 min per stage), and then 100% (three times and 30 min each time). The dehydrated samples were then infiltrated in stages with spur resin–ethanol solutions containing 50%, then 75%, and then 100% resin (1 h per stage). The infiltrated samples were left in 100% spur resin overnight. Next, the samples were embedded in the models with fresh resin and were polymerized at 70 °C for 24 h. The embedded cells were cut using an ultramicrotome (Leica Biosystems, Wetzlar, Germany) into 70-nm ultrathin sections by a diamond knife and were harvested on nickel grids. The grids were then post-stained with uranyl acetate and lead citrate. Transmission electron microscopy was carried out on a JEM-1400 (JEOL, Tokyo, Japan) at 120 keV equipped with a CCD camera system (Ultrascan, Los Angeles, CA, USA).
3
Preparing Bone Samples for TEM Analysis
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Marrow was flushed from the tibia after removing the epiphyses, and the bone fixed in 0.1 mol/L sodium cacodylate, pH7.4, containing 4% paraformaldehyde, 2.5% glutaraldehyde and 8.0 mmol/L CaCl2 at 4°C overnight, followed by decalcification with 14% EDTA for a week, as described previously.45 The shafts were trimmed to 1 mm length, postfixed with 1% osmium tetroxide, stained with 1% tannic acid and 0.5% uranyl acetate, and dehydrated in an ethanol series followed by propylene oxide. The samples were infiltrated and embedded in a mixture of Embed812 (Electron Microscopy Sciences), Araldite, dodecenylsuccinic anhydride, and DMP‐30. One hundred‐nm sections were cut with a DiATOME blade (Electron Microscopy Sciences) using an ultramicrotome (Leica Biosystems). The cross‐sections were adhered to copper grids (G100H‐Cu, Electron Microscopy Sciences) and examined at 80 kV using a transmission electron microscope (FEI Tecnai F20) equipped with a digital camera (FEI 4k Eagle).
4
Ultrastructural Analysis of Cockroach Thoracic Muscles
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P. americana thoracic muscles were dissected in Ringer saline and immediately fixed in 2.5% glutaraldehyde (electron microscopy grade) in 0.2 M phosphate buffer at 4 °C for 7 days. After fixation, the muscles were washed three times for 15 min with 0.1 M phosphate buffer (pH 7.4). Then, the samples were post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h for better ultrastructure preservation and contrast. The samples were washed again, dehydrated in a series of acetone solutions, and embedded into Epon-Araldite resin. Ultrathin sections were cut using an ultramicrotome (Leica Biosystems, Wetzlar, Germany) with diamond knives (Diatome Ltd., Nidau, Switzerland) and examined under a JEOL JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). Images were captured using a Sis MegaView III CCD camera (EMSIS, Münster, Germany).
5
Comprehensive Histological Analysis of Fixed Tissues
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Fixed tissue was watered with tap water, dehydrated in ascending ethanol (JT Baker, Phillipsburg, USA), and embedded in paraffin wax (Carl Roth, Karlsruhe, Germany). Samples were cut with an ultramicrotome (4 μm sections) (Leica Biosystems, Wetzlar, Germany). Afterward, standard histological staining, including haematoxylin and eosin (H&E) staining, iron staining (Prussian Blue), cresyl violet staining (Nissl staining), and elastic staining (Verhoeff Van Gieson / EVG) was performed. Slides were analysed with an Olympus BX46 (Olympus, Tokyo, Japan) microscope, and pictures recorded using an Olympus XC50 camera (Olympus, Tokyo, Japan). Additionally, immune fluorescence staining for GFAP and Tubulin was performed using antibodies and the protocol described above.
6
Ovarian Ultrastructure Analysis of Postnatal Mice
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The ovaries of postnatal-day-4 wild-type (WT) female mice were fixed overnight using 2% paraformaldehyde and 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (PB, pH 7.4) at 4°C. Following fixation, the samples were washed three times with 0.1 M PB for 30 min, and fixed with 2% osmium tetroxide in 0.1 M PB for 2 h at 4°C. Samples were dehydrated in graded ethanol solutions (50%: 20 min at 4°C; 70%: 20 min at 4°C; 90%: 20 min at RT; 100%: 20 min at RT), infiltrated twice with propylene oxide (PO) for 30 min, and added to mixed solutions of 70% PO and 30% resin (Nisshin EM Co.) for 1 h. Sample tube caps were left open overnight to evaporate the volatile PO, before the samples were transferred into 100% resin and polymerized for 48 h at 60°C. Polymerized resin samples were cut into 70-nm sections using a diamond knife and ultramicrotome (Leica Biosystems). The sections were then stained with 2% uranyl acetate for 15 min at RT, washed with distilled water, and then stained with lead stain solution (Sigma-Aldrich Corporation) for 3 min at RT. The stained sections were analyzed using a transmission electron microscope (JEOL Ltd), and their images were captured using a charge-coupled device camera (JEOL Ltd).
7
Ultrastructural Analysis of Oocyte Maturation
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The ovaries of 10-week-old Oog1pro3.9 mice were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M PB (pH 7.4) for 1 h at 4°C, after which they were washed three times for 15 min each in 0.1 M PB. The fixed ovaries were then dehydrated in 50% and 70% ethanol solutions for 30 min at 4°C before being infiltrated three times with a mixture of ethanol and resin (1:1) for 30 min at 4°C. Samples were then transferred to fresh 100% resin and subjected to ultraviolet polymerization overnight at 4°C. The ovaries embedded in polymerized resin were sliced into ultrathin 80-nm sections using a diamond knife and ultramicrotome (Leica Biosystems) and mounted on nickel grids. Sections were then incubated overnight at 4°C with rabbit anti-GFP antibody (Abcam, Inc.) in PBS containing 1% bovine serum albumin (BSA), washed three times for 1 min each with 1% BSA/PBS, and incubated with anti-rabbit IgG antibody conjugated with 10 nm gold particles for 2 h at RT. Following the three PBS washes, the sections were fixed in 2% glutaraldehyde in 0.1 M PB, dried, and then were stained for 15 min with 2% uranyl acetate. Lastly, the sections were stained with lead stain solution (SIGMA-Aldrich Corporation) for 3 min at RT, and images of the stained sections were captured using the JEM-1400plus transmission electron microscope (JEOL Ltd).
8
Transmission Electron Microscopy of A549 Cells
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A549 cells (2 × 105 cells/well) were grown on a six-well plate for 24 h, followed by 24 h of 4 μM Smp43 co-incubation. Cells without Smp43 treatment were considered the negative control. The harvested A549 cells were re-suspended in the TEM fixative, and then fixed at 4 °C for 2 h. Subsequently, the cells were collected via centrifugation, the supernatants were discarded, and the precipitations were washed with 0.1 M PB (pH = 7.4) three times for 3 min. A concentration of 1% of a prepared agarose solution was added for pre-embedding. After being fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature, samples were rinsed in 0.1 M PB (pH 7.4) 3 times, 15 min each, followed by being dehydrated at room temperature with a gradient of ethanol. Samples were inserted into the pure EMBed 812 (SPI, West Chester, IL, USA), and the removed resin blocks from the embedding models were cut until they were 60–80 nm thin on the ultra-microtome (Leica Biosystems, Nussloch, Germany) and fished out onto the 150-mesh cuprum grids with formvar film. After being stained with a 2% uranium acetate-saturated alcohol solution and 2.6% lead citrate, the cuprum grids were observed under a TEM (Hitachi, Tokyo, Japan).
9
Casparian Band Visualization in Chinese Fir
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Primary roots of 6-week-old Chinese fir plants, which were approximately 8-10 cm long, were divided into three zones: zone I (0-1 cm), zone II (1-4 cm) and zone III (4-8 cm), and 0.3-cm segments were cut at 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 cm from the root tip. The samples were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 24 h at 4 °C. The samples were then rinsed in 0.1 M cacodylate buffer, dehydrated in a graded ethanol and acetone series, then infiltrated and embedded in Spurr resin (Electron Microscopy Sciences). The resin blocks were polymerized for 24 h at 70 °C, then cut into 10-µm slices using an ultramicrotome (Leica Biosystems). The Casparian bands were stained with 0.1% (w/v) berberine hydrochloride for 1 h and counter-stained with 0.5% (w/v) aniline blue for an additional hour, as reported previously (Brundrett et al. 1988; Man et al. 2018) . The stained sections were viewed under a Leica TCS SP8 confocal laser microscope (Leica Microsystems) with an excitation wavelength of 488 nm and a detection wavelength of 520 nm.
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