Cells were harvested, pelleted and fixed in paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95–100% alcohol) and embedded in Epon-Araldite resin (Canemco, St. Laurent, QC, Canada; 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope (Philips, Eindhoven, The Netherlands). Values for the area occupied by autophagic vacuoles and the cytoplasm were obtained with Image Pro Plus version 3 (Media Cybernetics, Silver Spring, MA, USA) and used for the calculation of the cytoplasmic area occupied by the autophagic vacuoles.
Ultramicrotome
Manufactured by Philips
Sourced in United States
The Ultramicrotome is a precision instrument used for cutting extremely thin sections of materials for analysis under an electron microscope. It is designed to produce uniform, high-quality samples with thicknesses ranging from several nanometers to a few micrometers. The core function of the Ultramicrotome is to provide a reliable and controlled method for sectioning a wide variety of materials, including biological tissues, polymers, and inorganic materials.
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9 protocols using ultramicrotome
1
Ultrastructural Analysis of Autophagy
2
Ultrastructural Analysis of Autophagic Vacuoles
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Cells were fixed in 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95–100% alcohol) and embedded in Epon-Araldite resin (Canemco, 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope. The cells with autophagic vacuoles were defined as cells that had 5 or more autophagic vacuoles60 (link). Values for the area occupied by autophagic vacuoles and the cytoplasm were obtained with Image Pro Plus version 3.
3
Transmission Electron Microscopy of Autophagic Vacuoles
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Transmission electron microscopy analysis was done as described previously8 (link). Briefly, cells were fixed in 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed and ultimately stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95–100% alcohol) and embedded in Epon-Araldite resin (Canemco, 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope. The cells with autophagic vacuoles were defined as cells that had 5 or more autophagic vacuoles. Values for the area occupied by autophagic vacuoles and the cytoplasm were obtained with Image Pro-Plus version 4.
4
Transmission Electron Microscopy of Autophagic Vacuoles
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Cells were harvested, pelleted and fixed in paraformaldehyde (0.1% glutaraldehyde in 0.1 M sodium cacodylate) for 2 h, postfixed with 1% OsO4 for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95–100% alcohol) and embedded in Epon-Araldite resin (Canemco, 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope. Cells with autophagic vacuoles were defined as positive if they had 5 or more autophagic vacuoles. The area occupied by autophagic vacuoles and the cytoplasm were determined with Image Pro Plus Image Analysis Software version 3 and used to calculate the cytoplasmic area occupied by the autophagic vacuoles [50] (link).
5
Ultrastructural Changes in Ischemic BBB
6
Ultrastructural Analysis of Caco-2 Cells
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After F. nucleatum infection, Caco-2 cells were fixed in a solution containing 0.1% glutaraldehyde, 2% paraformaldehyde and 0.1 M sodium cacodylate for 2 hours, and fixed with 1% OsO4 for another 2 hours, washed three times with 1ml PBS and stained in 3% aqueous uranyl acetate for 1 hour. The fixed Caco-2 cells were then washed three times again, dehydrated with a graded alcohol series (40%, 50%, 70%, 80%, 90% and 100%), and finally embedded in Epon-Araldite resin (Canemco, #034). Ultrathin sections were cut with a Reichert ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 electron microscope.
7
Electron Microscopy Tissue Preparation
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Cells were harvested and fixed in a solution containing 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed and stained in 3% aqueous uranyl acetate for 1 h. The samples were then washed again, dehydrated with a graded alcohol series, and embedded in Epon-Araldite resin. Ultra-thin sections were cut on a Reichert ultramicrotome, counter-stained with 0.3% lead citrate and examined on a Philips EM420 electron microscope.
8
Ultrastructural Analysis of Mouse Kidney
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Freshly prepared kidney tissues of mice were immediately fixed for 3 days in 2.5% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer (pH = 7.4) and post-fixed in 1% OsO4, followed by washing in distilled water and en bloc staining in 3% uranyl acetate. Dehydration was conducted using a 70–100% graded ethanol. The tissues were then embedded in Epon polymer and ultra-thin sections (70 nm) were obtained with a diamond knife on a Reichert Jung ultramicrotome, stained with uranyl acetate-lead citrate and observed with a Philips CM-100 transmission electron microscope (FEI instruments, Hillsborough, OR, USA) at an accelerating voltage of 60 kV. The TEM images with magnifications ranging from 130,000× were analyzed for the examination of the morphology of subcellular constituents and structures.
9
Ultrastructural Analysis of Microglia
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Microglia were collected and fixed in a solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 hrs, postfixed with 1% OsO4 for 1.5 hrs, washed and stained in 3% aqueous uranyl acetate for 1 h. The samples were then washed again, dehydrated with a graded alcohol series, and embedded in Epon-Araldite resin (Canemco, #034). Ultrathin sections were cut on a Reichert ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 electron microscope.
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