To perform TEM, hICOs were fixed in 1.6% glutaraldehyde after treatment and stored at 4°C until processed. Subsequently, secondary fixation was performed using 1% osmium tetroxide for 1 hour. Next, acetone rinses were applied (8 times with an increased percentage of acetone for 15 minutes), and samples were infiltrated with 100% acetone/epoxy embedding medium (1:1) for 2 days. Samples were embedded for 1 hour at room temperature, followed by an embedding at 40°C overnight, and, finally, a 24-hour embedding at 60°C. Subsequently, ultrathin sections (60–70 nm) were created at a 6º angle at a speed of 1 mm/s with a Diatome Diamond knife (DiATOME, Hatfield, PA ) and Ultramicrotome Leica EM UC7 (Leica, Wetzlar, Germany). Finally, ultrathin sections were imaged using a Morgagni 268D microscope (FEI, Hillsboro, OR).
Morgagni 268d microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Morgagni 268D is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of samples. It features a 268 mm pole piece gap and can achieve a maximum magnification of 1,000,000x. The microscope is equipped with a LaB6 electron source and can operate at accelerating voltages up to 120 kV. It is suitable for a wide range of applications in materials science, nanotechnology, and biological research.
Automatically generated - may contain errors
6 protocols using morgagni 268d microscope
1
TEM Imaging of Hybrid Integrated Cells
2
Ultrastructural Analysis of Chordotonal Organs and Sperm Flagella
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine ultrastructure of chordotonal organs, legs from 36‐h‐old pupae were cut off with microscissors and fixed using a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/l sodium phosphate buffer, pH 7.2 for 2 h in a desiccator at room temperature and then overnight on a rotator at 4°C. Legs were then rinsed with sodium phosphate buffer, postfixed in 2% osmium tetroxide in buffer on ice, dehydrated in a graded series of acetone on ice, and embedded in Agar 100 resin. 70 nm sections were cut and poststained with 2% uranyl acetate and Reynolds lead citrate. Sections were examined with a Morgagni 268D microscope (FEI, Eindhoven, the Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus‐SIS).
To examine sperm flagellar ultrastructure, late pupal testes from third instar larvae were dissected in PBS and fixed using 2.5% glutaraldehyde in 0.1 mol/l sodium phosphate buffer, pH 7.2 for 1 h at room temperature. Samples were then rinsed with sodium phosphate buffer, postfixed in 2% osmium tetroxide in dH2O on ice, dehydrated in a graded series of acetone, and embedded in Agar 100 resin. 70 nm sections were then cut and processed as above.
To examine sperm flagellar ultrastructure, late pupal testes from third instar larvae were dissected in PBS and fixed using 2.5% glutaraldehyde in 0.1 mol/l sodium phosphate buffer, pH 7.2 for 1 h at room temperature. Samples were then rinsed with sodium phosphate buffer, postfixed in 2% osmium tetroxide in dH2O on ice, dehydrated in a graded series of acetone, and embedded in Agar 100 resin. 70 nm sections were then cut and processed as above.
3
Ultrastructural Analysis of C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L4-stage worms were fixed in 2.5% glutaraldehyde in 0.1 mol/liter sodium phosphate buffer, pH 7.4, overnight at 4°C. Samples were postfixed in 0.5–2% osmium tetroxide in the same buffer, washed in ddH2O, dehydrated in a graded series of ethanol, and embedded in Agar100 resin. 70-nm serial sections were poststained with aqueous uranyl acetate and lead citrate and examined with a Morgagni 268D microscope (FEI) equipped with an 11-megapixel Morada CCD camera (Olympus-SIS) and operated at 80 kV. At least five animals were examined per condition. For electron tomography, 200-nm sections were examined with a microscope (Tecnai G2 20; FEI) equipped with a CCD camera (Eagle 4k HS; FEI) and operated at 200 kV. Single axis tilt series were acquired using SerialEM (Mastronarde, 2005 (link)) by stepwise tilting the sample on average from −60° to +60° in 1° increments. Tomogram reconstruction with 10-nm gold beads as fiducials and model generation was performed using the IMOD software package (Kremer et al., 1996 (link)).
4
Scaffold Structure Density Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of each scaffold structure density was conducted using a technique that had previously been developed by the authors [52 ]. The analysis was performed using 3 samples of each of the Type 1 and 2 scaffolds (specified in Section 2.1 ) and of the scaffolds with encapsulated ASCs cultivated for 10 days under standard conditions in a CO2-incubator (under the following conditions: +37 °C; 5% CO2; absolute humidity). In order to conduct the analysis, a template (a hollow cylinder allowing the removal of a scaffold fragment with a diameter of 0.64 cm2) was used to separate a sample fragment from each cultivated cell scaffold at test points (1, 3, 6, and 10 days). These samples were examined using electron transmission microscopy with a Morgagni 268D microscope (FEI, Hillsboro, OR, USA). The resulting photomicrographs (20 photomicrographs for each type of scaffold at each test point; at 14,000× magnification) were processed using ImageJ software (version 1.50i). The analysis included a threshold binarization procedure. The image field was taken as 100%. The area of interest (the biopolymer part of the structure) and the background image (pore lumen) were selected at the image scanning stage. The percentages of the biopolymer part of the scaffold and of the pore lumen in the scaffold structure were assessed.
5
Chemical Fixation and Embedding of L4-Stage Worms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L4‐stage worms were prepared using chemical fixation following the protocol described in Serwas & Dammermann (2015 (link)). Worms were fixed in 2.5% glutaraldehyde in cytoskeleton buffer (CB, 100 mM methyl ester sulfonate, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose in ddH2O, pH 6.1) overnight at 4°C. Samples were then washed 3× in 1xCB and postfixed for 30 min in 0.5% osmium tetroxide in CB. Fixed worms were then washed 3× in CB and 1× in ddH2O. Finally, samples were dehydrated for 15 min each in 40, 60, 80%, 2× in 95%, and 3× in 100% acetone. Samples were embedded in Agar100 resin after fixation and dehydration. 70 nm serial sections were prepared onto Cu 100 mesh grids with Formvar film, then poststained with aqueous uranyl acetate and lead citrate, and examined with a Morgagni 268D microscope (FEI) equipped with an 11‐megapixel Morada CCD camera (Olympus‐SIS) and operated at 80 kV.
6
Fixation and Embedding of L4-Stage Worms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L4-stage worms were prepared by chemical fixation as previously described [63] . In brief, worms were fixed in 2.5% glutaraldehyde in cytoskeleton buffer (100 mM methyl ester sulfonate, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose in ddH2O, pH 6.1) overnight at 4°C. Samples were then washed 3x in the same buffer and post-fixed for 30min in 0.5% osmium tetroxide in buffer, washed 3x in buffer and 1x in ddH2O. Finally, samples were dehydrated for 15min each in 40%, 60%, 80%, 2x in 95% and finally 3x in 100% acetone. Samples were embedded in Agar100 resin after fixation and dehydration. 70 nm serial sections were then prepared, post-stained with aqueous uranyl acetate and lead citrate and examined with a Morgagni 268D microscope (FEI) equipped with an 11-megapixel Morada CCD camera (Olympus-SIS) and operated at 80 kV.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!