HIV-1 Gag-iFRET, -iFRETΔPro, or NL4-3 virions were produced as described in section “Cell Cultures and Virus Production” up to the viral pellet. The pellet was then fixed overnight at 4°C with a 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1M PBS solution. The next day, the pellet was washed twice with 0.1M PBS and post-fixed in 1% Osmium tetroxide (OsO4) for 1 h at room temperature (RT), then dehydrated in a series of graded ethanol solutions. After immersion in propylene oxide (Nacalai Tesque), samples were once again immersed in a mixture (1:1) of propylene oxide and LUVEAK-812 (Nacalai Tesque) overnight, embedded in Epon812 resin according to the inverted beam capsule procedure, and polymerized at 60°C for 2 days. Ultrathin sections were examined with an H-7650 electron microscope (Hitachi).
Propylene oxide
Manufactured by Nacalai Tesque
Sourced in Japan
Propylene oxide is a colorless, flammable liquid that is commonly used as an intermediate in the production of various chemicals and materials. It serves as a precursor for the synthesis of other compounds, playing a core role in chemical manufacturing processes.
Automatically generated - may contain errors
Lab products found in correlation
Osmium tetroxide, by Fujifilm (2 mentions)
H 7650 electron microscope, by Hitachi (1 mentions)
Luveak 812, by Nacalai Tesque (1 mentions)
H 7650 transmission electron microscope, by Hitachi (1 mentions)
Epoxy resin, by Nacalai Tesque (1 mentions)
Ultracut s, by Leica (1 mentions)
Bz 9000, by Keyence (1 mentions)
Glutaraldehyde, by TAAB (1 mentions)
Jem 1400ex, by JEOL (1 mentions)
Epon 812, by TAAB (1 mentions)
S 4700, by Hitachi (1 mentions)
5 protocols using propylene oxide
1
Ultrastructural Analysis of HIV-1 Virions
2
Ultrastructural Analysis of Intracranial Aneurysms
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At 5 days after the aneurysm induction, rats were killed as described previously. Dissected IA preparations were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Fixed specimens were then stained with 1% osmium tetroxide (Wako Pure Chemical Industries, Osaka, Japan) in 0.1 M sodium phosphate buffer (pH 7.4) for 1 h at room temperature. After dehydration in a series of graded ethanol solutions and replacement by propylene oxide (Nacalai Tesque), specimens were embedded in epoxy resin (Nacalai Tesque) overnight followed by the polymerization for 72 h. Ultrathin sections were prepared and images were obtained using an H‐7650 Transmission Electron Microscope (Hitachi, Tokyo, Japan).
3
Ultrastructural Analysis of Nerve Regeneration
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At 7 days after surgery, nerve-injured segments were harvested and fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment Ltd., Reading, Berkshire, United Kingdom) overnight at 4 °C. The nerve segments were subsequently fixed in 2% osmium tetroxide (OsO4; TAAB Laboratories Equipment Ltd.) for 2 h, separately dehydrated in an ethanol gradient (50%, 70%, 80%, 90%, 95% and 100%), and treated in a gradient of EPON812 (33%, 50%, 66% and 100%; TAAB Laboratories Equipment Ltd.) in propylene oxide (Nacalai Tesque, Inc., Kyoto, Japan). Tissues were embedded in EPON812 in a 60 °C oven for 48 h. Semi-thin sections (200 μm) were cut vertically with an ultramicrotome (Ultracut S; Leica Microsystems, Wetzlar, Germany), stained with 1% toluidine blue solution, and examined under a light microscope (BZ9000; Keyence). The density of the myelinated fibers (fibers/1000 mm2) was analyzed in five non-overlapping visual fields per specimen. Ultrathin sections (70–80 nm) were cut with an ultramicrotome. We chose axons exhibiting an equivalent diameter and evaluated the G-ratio as the ratio of the inner axonal diameter to the total outer diameter. The stained samples were observed under TEM (JEM-1400EX; JEOL Ltd., Tokyo. Japan). We randomly selected five separate fields per slice for analysis.
4
Ultrastructural Analysis of Immune-positive Reactions
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When immune‐positive reactions occur in the light microscopic studies of IHC‐N, it is the sign of a possibility of the development of IEM due to preservation of the intact ultrastructure, being free from protease digestion.12 , 13 , 46 The post‐fixed specimens of the liver were cut into 50‐μm‐thick sections with a Microslicer (Dosaka EM; Kyoto, Japan), which were then applied to a free‐floating procedure of the pre‐embedding method,12 , 13 , 46 in principally the same manner as was used for ICC for paraffin sections, except for the HRP‐labeled goat anti‐mouse IgG/Fab’ (MBL; Nagoya, Japan) used as the secondary antibody. The color‐developed specimens obtained were then fixed with 1.0% osmium tetraoxide in 50 mmol/L cacodylate buffer, pH 7.4, for 1 hour at RT and dehydrated in a series of graded ethanol solutions. After immersion in propylene oxide (Nakalai Tesque) (three times for ten min each), the samples were immersed in a mixture (1:1) of propylene oxide and Epon 812 resin (Taab Lab; reading Berks, UK) overnight and embedded in Epon 812 resin in a routine way. The regions to be studied were cut with a 2‐mm diameter punch, mounted to Epon blocks, and sectioned into ultrathin sections (LKB 8800 Ultrotome®III), which were then immediately observed in a 100CX Jeol electron microscope.
5
Scanning Electron Microscopy Sample Preparation
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The specimens were fixed with 2 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) and then stained with 1 % osmium tetroxide (Wako) in 0.1 M sodium phosphate buffer (pH 7.4). After the dehydration in a series of graded ethanol and the replacement by propylene oxide (Nacalai Tesque, Kyoto, Japan), the tissue was coated with a thin layer of platinum palladium using an ion sputter-coater (IB3, Eiko Corporation, Tokyo, Japan). Finally, images were obtained with a high-resolution scanning electron microscopy (S-4700, Hitachi High-Tech Corporation, Tokyo, Japan).
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