Scanning electronic microscopy (SEM) was performed as previously described with modifications (Du et al., 2020 (link)). Briefly, embryos after 100% epiboly were treated with 0.06–0.08 g/L PTU (P110661, Aladdin Industrial Corporation, China) to block pigment synthesis. Then larvae at 72 hpf were anesthetized with 0.17 mg/mL–1 Tricaine (MS-222, Sigma) for 30 s and fixed with 2.5% glutaraldehyde at 4°C over night, followed by post-fixation with 1% osmium tetroxide at 4°C for 2 h. After dehydration in ethanol, samples were critically point dried using a Leica EM CPD300 (Leica, Germany), then mounted and sputter coated with 10-nm platinum using a Cressington 108 sputter coater (Cressington, United Kingdom). Images were taken using a Quanta250 field-emission scanning electron microscope (FEI, Netherlands) with a beam strength of 3 kV.
Leica em cpd300
Manufactured by Leica camera
Sourced in Germany, Austria
The Leica EM CPD300 is a critical point dryer for electron microscopy sample preparation. It is designed to remove liquid from samples while preserving their structure and morphology. The device uses liquid carbon dioxide to transition samples from a liquid to a gaseous state, avoiding the damaging effects of surface tension that can occur during conventional drying methods.
Automatically generated - may contain errors
Lab products found in correlation
Sputter coater 108, by Cressington (5 mentions)
Quanta250 field emission scanning electron microscope, by Thermo Fisher Scientific (3 mentions)
Fe sem, by Zeiss (3 mentions)
Ace600, by Leica camera (3 mentions)
Quanta 250, by Thermo Fisher Scientific (2 mentions)
Leica scd500, by Leica camera (2 mentions)
Evo hd, by Zeiss (2 mentions)
Leica em ace600, by Leica camera (2 mentions)
Tm3000, by Hitachi (2 mentions)
S 4800, by Hitachi (2 mentions)
Ms 222, by Merck Group (1 mentions)
Phosphate buffer, by Merck Group (1 mentions)
Leica em scd500, by Leica camera (1 mentions)
4 well plate, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffer saline pbs, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Supra 55 feg sem, by Zeiss (1 mentions)
Leo 1530 gemini field emission scanning electron microscope, by Zeiss (1 mentions)
Em ace200, by Leica camera (1 mentions)
Jsm 6010plus la scanning electron microscope, by JEOL (1 mentions)
30 protocols using leica em cpd300
1
Zebrafish Larval SEM Imaging
2
Fixation and Freezing of Mating Pairs
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Copulations were initiated, as described above (s. recording of mating events), and a male was placed in the tray. Thirty seconds after the initiation of mating the couples were fixed with ca. 2 ml of 100% ethanol cooled to −80 °C and then frozen at −80 °C for 2 weeks. After thawing, the specimens were transferred into fresh 100% ethanol (1 h, three times alternatingly). Twelve pairs of S. ovinae fixed in copula were obtained. Three copulae were critical point dried, as well as a female with a broken penis in the cephalothoracic invagination, the corresponding male with missing penis, and a male with unfolded penis (Leica EM CPD300, Leica, Wetzlar, Germany).
3
Titanium Surface Sterilization and Cell Seeding
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To sterilize the titanium samples, they were autoclaved in water steams at 121 °C and a pressure of 1.5 atmospheres. Then, the samples were placed into the wells of 4-well plates (Nunc, USA). FetMSCs and MG-63 cells were seeded (1 × 105 in 20 µL of DMEM/F12 nutrient medium) on the surface of the samples and maintained for 24 h in CO2. After incubation, the cells were washed with Dulbecco’s phosphate buffer saline (PBS) (Sigma-Aldrich, Burlington, MA, USA) and fixed in 2.5% glutaraldehyde in phosphate buffer (pH = 7.2, Sigma-Aldrich, Burlington, MA, USA). After 3 days, the samples were washed in a phosphate buffer and successively dehydrated in 30, 50, 70, 90, 96% and absolute ethanol for 30 min each. The final drying was carried out 3 times for 15 min using Leica EM CPD300 at the CO2 critical point. Finally, the Au coatings with a thickness of about 10 nm were deposited with Leica EM SCD500. The evaluation of the cells’ morphology was performed using a Tescan MIRA3 LMU scanning electron microscope (TESCAN, Brno–Kohoutovice, Czech Republic).
4
Imaging Leaf Surface Morphology
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Transgenic and WT plants were grown in a greenhouse for four weeks, and then the features of the adaxial and abaxial surfaces in the same regions were observed during the mature leaf stage. Leaves were immediately fixed with 2.5% (v/v) glutaraldehyde for 2.5 h. After rinsing three times with 0.1 mol L−1 phosphoric acid buffer, the leaves were fixed in 1% (v/v) osmic acid for 2 h, followed by dehydration with the LEICA EM CPD 300 (Leica, Wetzlar, Germany) critical point dryer. EIKO IB-3 (Variable Electron Microscope, S3400N; Hitachi, Tokyo, Japan) was used to sputter gold on each sample, and surface images of the leaves were collected using a scanning electron microscope.
5
Ultrastructural Analysis of Bacterial Conjugation
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The ultrastructure of conjugation in all treatments was evaluated by scanning microscopy, using a modified method [89 (link)], considering the growth conditions in Luria Bertani broth (LB-Neogen®) and conjugation at 37 °C for 3 h/20 rpm. After conjugation, samples were fixed on 1-cm2 (PU) slides (Habasit Cleandrive TM, Reinach, CH) and fixed with 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M PBS buffer (pH 7.4) at 4 °C overnight. Subsequently, the fixative was removed, and the samples were washed with PBS buffer. The samples were post-fixed for 2 h with 1% osmium tetroxide and washed three times with PBS buffer. The dehydrated series was applied with ethanol solutions (30, 40, 50, 60, 70, 80, and 90% and then three times at 100%) for 20 min for each step. The samples were dried in a CPD (critical drying point) system (Leica EM CPD300; Leica, Wien, Austria) using liquid carbon dioxide as a transition fluid, coated with a 20-nm gold layer (SCD 050, Baltec), and exposed on a Zeiss Supra 55 FEG SEM operating at 5 kV. Images were captured with standard ZEISS SmartSEM.
6
Scanning Electron Microscopy of Microbes
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Herein, 1 mL of microbial suspension (1 × 105 cells/mL) was placed onto a 24-well plate and incubated at 37 °C for 24 h. For microscopic examination, the microbe was placed in 2% paraformaldehyde–glutaraldehyde in 0.1 M PBS for at least 30 min at room temperature. The samples were postfixed with 1% OsO4, which was dissolved in 0.1 M PBS for 2 h. Thereafter, they were dehydrated in an ascending gradual ethanol series, treated with isoamyl acetate, and subjected to a critical point drying process (LEICA EM CPD300; Leica, Wien, Austria). Subsequently, the samples were coated with Pt (5 nm) using an ion coater (ACE600; Leica) and examined and photographed using a scanning electron microscope (FE-SEM; Merin, Carl Zeiss, Oberkochen, Germany) at 2 kV.
7
Scanning Electron Microscopy of Bacteria
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Bacteria were fixed with glutaraldehyde (2.5% [vol/vol]) for 1 h at room temperature while sedimenting on poly-l -lysine-coated coverslips. After two washings in cacodylate buffer (100 mM, pH 7.2), cells were dehydrated by increasing concentrations of ethanol, followed by critical-point drying in a Leica EM CPD300 automated critical-point dryer (Leica, Germany). Then, the coverslips were mounted on aluminum sample holders (stubs) and gold sputter coated (layer thickness, 20 nm) in a BAL-TEC SCD 005 sputter coater (BAL-TEC, Liechtenstein). Images were acquired with a Zeiss (LEO) 1530 Gemini field emission scanning electron microscope (Zeiss AG, Germany) at a 4-kV acceleration voltage and a working distance of 3 to 4 mm using an Inlense secondary electron detector.
8
Scanning Electron Microscopy of Parasites
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Parasites were washed twice in PBS and fixed for 1 h at room temperature in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. The fixed parasites were washed twice in 0.1 M cacodylate buffer and then adhered for 15 min at room temperature to 0.1% poly-L-lysine coated coverslips. The non-adhered cells were washed out twice with 0.1 M cacodylate buffer and the adhered cells were then incubated for 15 min at room temperature with 1% osmium tetroxide diluted in 0.1 M cacodylate buffer. The samples were washed three times with 0.1 M cacodylate buffer and dehydrated in a graded acetone series (30%, 50%, 70%, 90% and 100% acetone, 5 min each). This step was followed by critical point drying in a Leica EM CPD300 and gold sputtering in a Leica EM ACE200. The samples were visualized in a JEOL JSM-6010 PLUS/LA scanning electron microscope at 20 kV.
9
Scanning Electron Microscopy of Mouse Cochlea
10
SEM Characterization of Gelled ECM
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