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ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin...

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Detalles Bibliográficos
Autores principales: Bernhard, W., Leduc, Elizabeth H.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1967
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2107175/
https://www.ncbi.nlm.nih.gov/pubmed/4167504
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author Bernhard, W.
Leduc, Elizabeth H.
author_facet Bernhard, W.
Leduc, Elizabeth H.
author_sort Bernhard, W.
collection PubMed
description A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.
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spelling pubmed-21071752008-05-01 ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation Bernhard, W. Leduc, Elizabeth H. J Cell Biol Article A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies. The Rockefeller University Press 1967-09-01 /pmc/articles/PMC2107175/ /pubmed/4167504 Text en Copyright © 1967 by The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Bernhard, W.
Leduc, Elizabeth H.
ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title_full ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title_fullStr ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title_full_unstemmed ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title_short ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation
title_sort ultrathin frozen sections : i. methods and ultrastructural preservation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2107175/
https://www.ncbi.nlm.nih.gov/pubmed/4167504
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