We examined the characteristics of degranulated eosinophils using EM. Two pieces of extracted tissue were pre-immobilized for 2 hours on 2.5% glutaraldehyde adjusted with 0.1 M phosphate buffer solution (pH 7.4) and then washed with the same buffer solution 3 times for 30 minutes each. After 1 hour, the tissue was immobilized on 1% osmium tetroxide (0.1 M phosphate buffer solution, pH 7.4). The post-mobilized sample was washed with the same buffer solution, dehydrated in ethanol, which was substituted with propylene oxide twice for 15 minutes each, and embedded with Epon812. The embedded sample was polymerized and cut cross-sectionally at a 1 μm thickness using an ultramicrotome (Reichert Technologies, Buffalo, NY, USA), and the slice was stained with 1% toluidine blue. The regions infiltrated with eosinophils were identified by optical microscopy, and 60–70 nm ultrathin sections were prepared and double stained. The sections were examined by EM using the H-7650 Hitachi microscope (Tokyo, Japan). Figure 3 shows an electron micrograph in which the core matrix density of the secondary eosinophil granules changed and became transparent.
Ultramicrotome
Manufactured by Reichert Technologies
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The Ultramicrotome is a precision instrument designed for sectioning samples in preparation for electron microscopy. It utilizes a diamond knife to cut extremely thin sections, typically less than 100 nanometers in thickness, from a sample embedded in a resin block. The Ultramicrotome allows for the production of high-quality, ultrathin sections required for detailed analysis and visualization of microscopic structures.
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Lab products found in correlation
H 7650, by Hitachi (9 mentions)
Buffered 1 osmium tetroxide, by Polysciences (8 mentions)
Diamond knife, by Electron Microscopy Sciences (7 mentions)
Cryosubstitution unit, by Leica (4 mentions)
Lowicryl resin, by Electron Microscopy Sciences (4 mentions)
Formvar carbon coated nickel slot grids, by Electron Microscopy Sciences (4 mentions)
Jem 1010, by JEOL (4 mentions)
Uranyl acetate, by Electron Microscopy Sciences (4 mentions)
Jem 1011, by JEOL (4 mentions)
Formvar supported slot grid, by Electron Microscopy Sciences (3 mentions)
Lead citrate, by Thermo Fisher Scientific (3 mentions)
Epon mixture, by Polysciences (3 mentions)
Osmium tetroxide, by Polysciences (3 mentions)
Jem 1230, by JEOL (3 mentions)
Sigma500 transmission electron microscope, by Zeiss (3 mentions)
Em420, by Philips (2 mentions)
Vt1000, by Leica (2 mentions)
Karnovsky s fixative solution, by Merck Group (2 mentions)
Sigma 500, by Zeiss (2 mentions)
H 7000, by Hitachi (2 mentions)
95 protocols using ultramicrotome
1
Ultrastructural Analysis of Eosinophil Degranulation
2
Transmission Electron Microscopy Tissue Preparation
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The Karnovsky's fixed tissues were processed and embedded in Araldite CY212 to make blocks. Thin sections were cut (approximately 70-80 nm thickness) using an ultra-microtome (Reichert Technologies). Thereafter, the tissues were stained with uranyl acetate and lead acetate. These sections were visualised under a transmission electron microscope (Morgagni 268D; FEI company) operated at 80 kV. The sections were evaluated by a cytologist blinded to the study groups.
3
Quantitative Cell Area Analysis
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Sepals and fruits from wild type and transgenic plants were cut into little pieces that were subsequently fixed in 3 % glutaraldehyde in 0.1 M cacodylate buffer (pH 6.9) for 24 h and post-fixed for 2 h in 1 % osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a graded ethanol series and then embedded in araldite resin. Thin sections (1 μm), obtained with a Reichert-Jung ultramicrotome, were stained with 1 % toluidine blue for light microscopy.
By using the imaging software Leica Application Suite associated to the Leica DM 5000 B automated upright microscope, quantitative analyses were performed. In particular it was measured the cell area of 100 cells/each sample in1 μm thick sections obtained from three different control and transgenic fruits or sepals, respectively. The obtained values were expressed as the mean ± SE of 3 independent replicates for each sample, using the Student’s t-test to analyze the differences between the control and transgenic groups. Statistical significance was set at P < 0.05.
By using the imaging software Leica Application Suite associated to the Leica DM 5000 B automated upright microscope, quantitative analyses were performed. In particular it was measured the cell area of 100 cells/each sample in1 μm thick sections obtained from three different control and transgenic fruits or sepals, respectively. The obtained values were expressed as the mean ± SE of 3 independent replicates for each sample, using the Student’s t-test to analyze the differences between the control and transgenic groups. Statistical significance was set at P < 0.05.
4
Transmission Electron Microscopy of Liver Samples
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Routine methods to prepare liver specimens for transmission electron microscopy were performed using at least 3 animals from each group. Fragments of liver tissue (1 mm 3 ) were fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room temperature [32] . HepG2 cells were collected and prepared as described previously [30] . Briefly, cells were fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate overnight, post-fixed with 1% osmium tetroxide for 1.5 h, washed, and stained for 1 h in 3% aqueous uranyl acetate. Ultrathin sections were cut on an ultramicrotome (Reichert-Jung, Inc., Cambridge, UK) and counterstained with 0.3% lead citrate. The tissue and cell sections were examined by a transmission electron microscope (model no. EM420; Koninklijke Philips Electronics N.V., Amsterdam, The Netherlands).
5
Echinopsis terscheckii Floral Nectary Ultrastructure
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Floral nectary ultrastructure of Echinopsis terscheckii was studied in order to elucidate the probability of mixed secretion in the genus (through stomata and through epidermal trichomes). Floral nectary at pre-anthesis and anthesis were pre-fixed in 1% glutaraldehyde, 4% formaldehyde in phosphate buffer (pH 7.2) for 48 h and then post-fixed in OsO 4 at 2°C in the same buffer for 3 h. Then, they were dehydrated in ascending ethanol series and embedded in Spurr's resin (O'Brien & McCully 1981) . Sections 1 μm thick were made on a Reichert-Jung ultramicrotome and stained with toluidine blue. Ultrathin sections (750-900 nm) were stained with uranyl acetate and lead citrate (Zarlavsky 2014) . The sections were examined under a JEOL-JEM 1200 Ex II transmission electron microscopy (TEM) at 85 kV.
6
Ultrastructural Analysis of Parasites
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Parasites were washed in cold 100 mM sodium cacodylate buffer (pH 7.2) and suspended in the same buffer containing 2.5% (v/v) glutaraldehyde. Cells were fixed for 2 hours at room temperature, followed by post-fixation in 2% OsO4 in cacodylate buffer (pH 7.3). After three washes in distilled water, samples were pre-stained in saturated uranyl acetate solution in distilled water for 30 min at room temperature and extensively washed in distilled water. Dehydration was carried out by stepwise incubation in ethanol (30–50–70–90–100%). Parasites were then embedded in EPON812 resin as described previously59 . Sections (80 nm) were cut on an ultramicrotome (Reichert & Jung, Vienna, Austria), placed onto 300 mesh formvar-carbon-coated nickel grids (Plano, Wetzlar, Germany), and sections were stained with uranyl acetate and lead citrate60 (link). Specimens were viewed on a CM12 transmission electron microscope operating at 60 kV.
7
Transmission Electron Microscopy of CdS-Dx/QD Uptake in Cells
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HeLa and HEK293 cells grown in Tc-treated culture dishes (60 mm in diameter) were incubated with fresh 1 μg/ml CdS-Dx/QDs for 24h. Extracellular CdS-Dx/QDs were discarded after thorough washing with PBS. Processing for TEM was done according to Muñiz et al. [45 ]. Briefly, cells were fixed with 2.5 % glutaraldehyde for 2 h at RT, followed by thorough washings with PBS. Cells were scrapped off and then centrifuged at 2000 rpm to form a cell pellet. Cells were postfixed for 1 h in 1 % OsO4 at 4 °C. Samples were rinsed with PBS and then gradually dehydrated in increasing concentrations of ethanol and finally embedded in Spurr’s resin and polymerized at 60 °C for 48 h (Electron Microscopy Sciences, Washington, DC). Thin sections mounted on grids were obtained using a Reichert Jung ultramicrotome (Reichert Jung, Austria). They were afterwards stained with uranyl acetate and lead citrate. Grids were viewed in the TEM.
8
Ultrastructural Analysis of Biological Samples
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The specimen were cut into small parts and then immersed in 2.5% glutaraldehyde in PBS (4°C, pH 7.4, 0.1 M) for overnight. Tissue were rinsed in the same PBS and then post‐fixed for 60 min. at room temperature by using buffered 1% osmium tetroxide (Polysciences Inc., Warrington, PA, USA) and washed in the buffer. The samples were then dehydrated in ascending concentrations of ethyl alcohol, infiltrated with a propylene oxide–Araldite mixture and then embedded in Araldite. The blocks were then sectioned by using an ultramicrotome (ReichertJung, Wien, Austria) and the ultrathin sections (50 nm) were mounted on cooper coated grids. The pieces were stained with 1% uranyl acetate and Reynold's lead citrate for 20 min. Finally samples were examined and photographed by using a high resolution digital camera (16 mega pixel) connected to the TEM, Hitachi H‐7650 (Toyko, Japan).
9
Ultrastructural Analysis of Medial Prefrontal Cortex
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Rats were anesthetized using 30% chloral hydrate and intracardially perfused with 1% paraformaldehyde (PFA) in phosphate buffer (PB, pH 7.4) for 2 min, followed by a xative comprising 4% PFA and 0.125% glutaraldehyde in PB for 10 min. The brains were immediately dissected, post-xed overnight at 4°C in 4% PFA and 0.125% glutaraldehyde in PB, and then transferred to phosphate buffered saline with 0.1% sodium azide at 4°C until sectioned. The brains were hemisected and the right hemisphere was cut into 250 ∝m-thick sections using a vibratome (VT1000S, Leica Microsystems, Buffalo Grove, IL). The sections were cryoprotected in a graded glycerol/PB solution after which the mPFC was microdissected into blocks that underwent cryosubstitution and low-temperature embedding as described earlier (57) . Five consecutive ultrathin sections were cut at 90 nm using a diamond knife (Diatome, Hat eld, PA) on an ultramicrotome (Reichert-Jung, Depew, NY) and mounted onto a Formvar-supported slot grid (Electron Microscopy Sciences, Hat eld, PA).
10
Hippocampal CA1 Region Ultrastructural Analysis
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Coronal sections encompassing the CA1 region of the hippocampus were prepared for EM as reported previously9 (link),33 (link),37 (link). Briefly, slices were cryoprotected in graded PBS/glycerol washes at 4 °C, and manually microdissected to obtain blocks containing the CA1 region. The blocks were rapidly freeze-plunged into liquid propane cooled by liquid nitrogen (− 190 °C) in a universal cryofixation system KF80 (Reichert-Jung, Depew, NY) and subsequently immersed in 1.5% uranyl acetate dissolved in anhydrous methanol at − 90 °C for 24 h in a cryosubstitution unit (Leica). Block temperatures were raised from − 90 to − 45 °C in steps of 4 °C/hour, washed with anhydrous methanol, and infiltrated with Lowicryl resin (Electron Microscopy Sciences, Hatfield, PA) at − 45 °C. The resin was polymerized by exposure to ultraviolet light (360 nm) for 48 h at − 45 °C followed by 24 h at 0 °C. Block faces were trimmed and ultrathin Sects. (90 nm) were cut with a diamond knife (Diatome, Hatfield, PA) on an ultramicrotome (Reichert-Jung) and serial sections were collected on formvar/carbon-coated nickel slot grids (Electron Microscopy Sciences).
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