For transmission electron microscope (TEM) analysis, rats were initially perfused with a 0.1M PO4 solution (pH 7.3) followed by a 2% glutaraldehyde/4% paraformaldehyde solution in 0.1M PO4 with pH set at 7.3. Neural lobes were excised en toto and post-fixed overnight, followed by 4% OsO4 for 60 minutes at 4°C (Ted Pella). NL’s were then dehydrated in increasing concentrations of ethanol, cleared in propylene oxide (Ted Pella), then vacuum infiltrated in a 100% Epon/Araldite mixture (50:50, v/v; Ted Pella), overnight. Subsequent polymerization was performed at 58°C for 72 hrs. Coronal 1 mm serial sections of the intact NL were collected from a rostral to caudal direction and all NL samples were mounted on formvar-coated slot grids, then contrast stained with Reynold’s lead citrate uranyl acetate in an LKB Ultrastainer and viewed on a Zeiss TEM 10C at 60 kV. Nine sampled fields from each of three intact NL cross sections were collected at 5000X.
Propylene oxide
Manufactured by Ted Pella
Sourced in United States
Propylene oxide is a colorless, volatile, flammable liquid used as a chemical intermediate in the production of various industrial and consumer products. It is a versatile compound that serves as a key starting material for the synthesis of other chemicals and materials.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glutaraldehyde, by Ted Pella (1 mentions)
Pipes, by Merck Group (1 mentions)
Epon resin, by Ted Pella (1 mentions)
Propylene oxide, by TAAB (1 mentions)
Ethanol, by Merck Group (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
1400 electron microscope, by JEOL (1 mentions)
Orius sc1000 ccd digital camera, by Ametek (1 mentions)
Lx112 resin, by Ted Pella (1 mentions)
3 protocols using propylene oxide
1
Ultrastructural Analysis of Rat Neural Lobes
2
Transmission Electron Microscopy Sample Preparation
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For transmission electron microscopy, the samples were fixed in 2.5% glutaraldehyde (Ted Pella INC, Redding, CA, USA) + 1% paraformaldehyde (Merck, Darmstadt, Germany) in PIPES (Merck, Darmstadt, Germany) pH 7.4, and stored at 4 °C until further processed. Samples were rinsed with 0.1 M phosphate buffer for 10 min prior to 1 h incubation in 1% osmium tetroxide (TAAB, Aldermaston, England) in 0.1 M phosphate buffer. After rinsing in 0.1 M phosphate buffer, samples were dehydrated by incubation in increasing concentrations of ethanol (50%, 70%, 95% and 99.9%) for 10 min each, followed by 5 min incubation in propylene oxide (TAAB, Aldermaston, England). The tissue samples were thereafter placed in a mixture of Epon Resin (Ted Pella INC, Redding, CA, USA) and propylene oxide (1:1) for 1 h, followed by 100% resin, and left over night. Subsequently, the samples were embedded in capsules in newly prepared Epon Resin, left for 1–2 h, and then polymerized at 60 °C for 48 h.
3
Ultrastructural Analysis of Ileum Tissue
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Samples were treated as previously described (Palmer et al., 2017 (link)). Tissues from the terminal ileum were cleared by flushing with HBSS (with calcium) at 37o C and then fixed by vascular perfusion with 2% glutaraldehyde in Gomori phosphate buffer containing 0.1 mM EGTA (pH 7.4) at 37°C. Tissues were excised and cut along the antimesenteric plane, pinned flat in fixative for 10 min at RT, cut into small pieces, and then fixed o/n at 4°C in the same fixative. Tissues were washed in PBS, postfixed in 1% osmium tetroxide in Gomori phosphate buffer pH 7.4 for 1 h at 4° C, washed in distilled water, and then incubated o/n in 2% aqueous uranyl acetate at 4°C. After washes with distilled water, tissues were dehydrated in ethanol (Sigma-Aldrich, Natick, MA) and propylene oxide (Ted Pella, Redding, CA) prior to resin embedding in LX112 resin (Ted Pella). Ultrathin sections were cut with a Leica Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany), placed on formvar and carbon-coated slot grids, and then contrast stained with 2% uranyl acetate and lead citrate. DMs from cells along two villi from each mouse were imaged using a JEOL 1400 electron microscope (JEOL USA, Peabody, MA) equipped with an Orius SC1000 digital CCD camera (Gatan, Pleasanton, CA).
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