Mice were deeply anesthetized with 10 % chloral hydrate and killed by transcardial perfusion with 1× PBS followed by 4 % paraformaldehyde in 1× PBS. Tissues were post-fixed in the same fixative overnight. Sciatic nerves were cryoprotected in 30 % sucrose in 1× PBS, and sectioned at 20 μm. Sciatic nerves were sectioned longitudinally using a Leica cryostat (model CM3050S; Leica Microsystems). For semithin sections, nerves, DRG, and spinal cords were postfixed in 4 % paraformaldehyde/2 % glutaraldehyde followed by OsO4 and embedded in Epon. Cross sections, at 1 μm thickness, were stained for toluidine blue and examined under light microscopy. For ultra-structural analyses, spinal cord and nerves were processed as described previously [24 (link)]. Briefly, 70 nm thin sections were obtained and stained for citrate/uranyl acetate. Electron micrographs were acquired using Zeiss Libra transmission electron microscope.
Libra transmission electron microscope
Manufactured by Zeiss
Sourced in United States
The Zeiss Libra transmission electron microscope (TEM) is a high-performance instrument designed for advanced materials analysis. It provides high-resolution imaging, diffraction, and spectroscopic capabilities for the study of a wide range of samples at the nanoscale level.
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5 protocols using libra transmission electron microscope
1
Tissue Processing for Ultrastructural Analysis
2
TEM Analysis of PMMA Nanoparticles
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Carbon/formvar-coated copper grids (200 #), used during the TEM studies, were purchased from Electron Microscopy Sciences PA, USA. TEM investigations were conducted using a ZEISS LIBRA Transmission Electron Microscope, operated at 120 keV. A drop of PMMA nanoparticles was taken on standard carbon formvar-coated copper grid (200 #), negatively stained with 2% magnesium uranyl acetate solution, for 30–60 s, and dried using 150 W lamp for 24 h. This procedure was conducted in a dust free zone, before recording the TEM. TEM analysis was performed on the PLGA55-coumarin-6 nanoparticles prepared with 0.375 mg/mL of the polymer.
3
Spectroscopic Characterization of Synthesized Compounds
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Deionized water was used to prepare the solutions. All chemicals were purchased from Sigma-Aldrich Company. Analytical solutions were prepared using metal ion nitrate salts. IR spectra were recorded by the Nicolet FT-IR NEXUS 670 spectrometer (Thermo Scientific, USA) and used to identify the presence of doped groups in synthesized compounds. Raman measurement was done by HANDHELD RAMAN ANALYZER of RIGAKU (FIRSTGUARD model). TESCAN MIRA III scanning electron microscope and Zeiss Libra transmission electron microscope (working at 100 kV) were used to record FE-SEM and TEM images respectively. XRD instrument of a Bruker D8 ADVANCE X-ray diffractometer was used with a Cu-Kα radiation source (λ = 1.5406 Å) operating at 40 kV, 40 mA, and a scanning range of 10–80° 2θ, with a 2θ scan step of 0.015° and a step time of 0.2 s. UV–Vis spectra were recorded by WPA Biowave LifeScience UV–Vis spectrometer using quartz cuvettes with a path-length of 10 mm in H2O as the solvent. pH measurements were performed using a digital pH meter (HANNA 212). Ultrasonic bath (KODO model JAC1002) was used to clean the surface of GCE and prepare homogeneous suspensions from modifiers.
4
Nerve Pathology in MLD Mouse Model
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Nerve pathology was examined in sciatic and urethral nerves from a male mouse model of aggravated MLD. This mouse model (transgenic[tg]/ASA[−/−]) possesses an ASA deficiency and overexpression of the sulfatide‐synthesizing enzyme galactose‐3‐O‐sulfotransferase‐1 in myelinating cells.
12 (link) Mice (two aggravated MLD mice and three control tg/ASA[+/−] mice; 16–20 months old) were fixed in a solution of 4% paraformaldehyde with 2% glutaraldehyde in 1× phosphate‐buffered saline. Sciatic nerves and penises were dissected out and post‐fixed in the same solution. The dissected samples were processed for electron microcopy analysis by immersion in osmium tetroxide followed by embedding in Epon (reagents from Electron Microscopy Sciences, PA, USA). The sciatic nerves and penises were sectioned at 70 nm and stained with citrate/uranyl acetate. Electron micrographs were acquired using a Zeiss Libra transmission electron microscope at 8000–10,000× magnification.
12 (link) Mice (two aggravated MLD mice and three control tg/ASA[+/−] mice; 16–20 months old) were fixed in a solution of 4% paraformaldehyde with 2% glutaraldehyde in 1× phosphate‐buffered saline. Sciatic nerves and penises were dissected out and post‐fixed in the same solution. The dissected samples were processed for electron microcopy analysis by immersion in osmium tetroxide followed by embedding in Epon (reagents from Electron Microscopy Sciences, PA, USA). The sciatic nerves and penises were sectioned at 70 nm and stained with citrate/uranyl acetate. Electron micrographs were acquired using a Zeiss Libra transmission electron microscope at 8000–10,000× magnification.
5
Visualizing Chloroplast Ultrastructure by Microscopy
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