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Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applicatio...

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Autores principales: Goldschlager, Tony, Abdelkader, Amany, Kerr, Jeffrey, Boundy, Ian, Jenkin, Graham
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152214/
https://www.ncbi.nlm.nih.gov/pubmed/20062000
http://dx.doi.org/10.3791/1707
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author Goldschlager, Tony
Abdelkader, Amany
Kerr, Jeffrey
Boundy, Ian
Jenkin, Graham
author_facet Goldschlager, Tony
Abdelkader, Amany
Kerr, Jeffrey
Boundy, Ian
Jenkin, Graham
author_sort Goldschlager, Tony
collection PubMed
description Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images(1) and allows for techniques such as fluorochrome assessment and histomorphometry(2). Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.
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spelling pubmed-31522142012-01-08 Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis Goldschlager, Tony Abdelkader, Amany Kerr, Jeffrey Boundy, Ian Jenkin, Graham J Vis Exp Medicine Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images(1) and allows for techniques such as fluorochrome assessment and histomorphometry(2). Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated. MyJove Corporation 2010-01-08 /pmc/articles/PMC3152214/ /pubmed/20062000 http://dx.doi.org/10.3791/1707 Text en Copyright © 2010, Journal of Visualized Experiments http://creativecommons.org/licenses/by/2.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Medicine
Goldschlager, Tony
Abdelkader, Amany
Kerr, Jeffrey
Boundy, Ian
Jenkin, Graham
Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title_full Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title_fullStr Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title_full_unstemmed Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title_short Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis
title_sort undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis
topic Medicine
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152214/
https://www.ncbi.nlm.nih.gov/pubmed/20062000
http://dx.doi.org/10.3791/1707
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