<p class="jove_content">The authors would like to acknowledge the assistance of Ms Sue Connell for her expertise in resin embedding and Ms Stephania Tombs for her laboratory expertise. The authors would like to thank Professor Frank Kandziora and Dr Marie-Anne Polboth for their advice.</p>

<ol>
  <li>The tissue is placed in a sealed container containing 10% phosphate buffered formalin solution<sup>3</sup>. Ensure minimal exposure to light, if fluorochrome analysis is to be performed, as the fluorochromes are light sensitive.   </li>
  <li>Establish which orientation of the tissue will be required. </li>
  <li>Trim the specimen to size, in the correct orientation, using a band saw. Ensure safety measures are adhered to. The specimen is then placed into an opaque container, the volume of which should be approximately ten times t...

<ol>
	<li>Kerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Atlas of Functional Histology</em>. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> <em style='font-style: italic'>J Bone Miner Res</em>. <b>2</b>, 595-610 (1987).</li><li>An, Y. H., Martin, K. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Handbook of histology methods for bone and cartilage</em>. , (2003).</li><li>Bancroft, J. D., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Theory and Practice of Histological Techniques</em>. , 352-360 (2008).</li></ol>

<u>Solvents:</u>
  <ul>
    <li>10% Normal Buffered Formalin, Ethanol, Xylene and Butanol acid<br />
      All histology grade, sulfur free.<br />Grale Scientific Pty Ltd.<br />http://www.grale.com.au</li>
</ul>
<u>Moulds:</u>
  <ul>
    <li>Peel away, plastic moulds<br />Grale Scientific</li>
</ul>
<u>Slides:</u>
  <ul>
    <li>SUPERFROST PLUS Microscope slides<br />Lomb Scientific Pty Ltd.<br />
  <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a></li>
  </ul>
<u>Resin:</u>
  <ul>
    <li>GlycolMethacrylate Technovit 7100<br />Heraeus Kulzer<br />
  <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a></li>
    <li>Backing Block Resin- Technovit 3040  Histo block<br />Heraeus Kulzer</li>
  </ul>
<u>Stains</u><br />
<span>(Reagents from Grale Scientific)</span>
<p>Goldner's Trichrome stain</li>
<p style="margin-left:40px;">1. Weigert's iron hemotoxylin (DeGroat)</p>
<table style="margin-left:40px;" border="0">
<tr><td colspan="2" style="font-weight:bold;">a) Hemotoxylin solution</td></tr>
<tr><td>Hemotoxylin</td><td>1g</td></tr>
<tr><td>Absolute Alcohol</td><td>100mL</td></tr>
<tr><td colspan="2"  style="font-weight:bold;">b) Iron Solution</td></tr>
<tr><td>30% aqueous ferric chloride</td><td>4mL</td></tr>
<tr><td>Hydrochloric acid (concentrated)</td><td>1mL</td></tr>
<tr><td>distilled Water</td><td>95mL</td></tr>
</table>
<p style="margin-left:40px;" >Filtered and added to equal volume of hemotoxylin immediately before stain used.</p>
<p style="margin-left:40px;" >2. Ponceau-fuchsin-azophloxin stock solutions</p>
<table style="margin-left:40px;" border="0">
<tr><td colspan="2"  style="font-weight:bold;">a) Ponceau de xylidine solution</td></tr>
<tr><td>Ponceau de xylidine</td><td>0.75g</td></tr>
<tr><td>acid fuchsion</td><td>0.25g</td></tr>
<tr><td>Acetic Acid</td><td>1mL</td></tr>
<tr><td colspan="2">mix, and add distilled water (100mL)</td></tr>
<tr><td colspan="2"  style="font-weight:bold;">b) azophloxin solution</td></tr>
<tr><td>Azophloxin</td><td>0.5g</td></tr>
<tr><td>Acetic acid</td><td>0.6mL</td></tr>
<tr><td colspan="2">mix, and add distilled water (100mL)</td></tr>
<tr><td colspan="2"  style="font-weight:bold;">c) Final working stain solution</td></tr>
<tr><td>Ponceau-fuchsin solution</td><td>5-10mL</td></tr>
<tr><td>Azophloxin</td><td>2mL</td></tr>
<tr><td>0.2% acetic acid solution</td><td>88mL</td></tr>
</table>
<p style="margin-left:40px;">3. Light Green Solution</p>
<table style="margin-left:40px;" border="0">
<tr><td>Light Green</td><td>1g</td></tr>
<tr><td>acetic acid</td><td>1mL</td></tr>
<tr><td colspan="2">Mix, and add distilled water (500mL)</td></tr>
</table>
<p style="margin-left:40px;">4. Phophomolybdic acid-Orange G solution</p>
<table style="margin-left:40px;" border="0">
<tr><td>Phosphomolybdic acid</td><td>3g</td></tr>
<tr><td>Orange G</td><td>2g</td></tr>
<tr><td colspan="2">Dissolve in 500mL of distilled water and add crystal thymol.</td></tr>
</table>
<p>Von Kossa Stain</p>
<table style="margin-left:40px;" border="0">
<tr><td>1% aqueous silver nitrate</td></tr>
<tr><td>2.5% sodium thiosulfate</td></tr>
<tr><td>1% safranin O</td></tr>
</table>
<p>Alcian blue stain</p>
<table style="margin-left:40px;" border="0">
<tr><td>Alcian blue 8GX</td><td>1g</td></tr>
<tr><td>3% acetic acid solution</td><td>100mL</td></tr>
</table>
<p>Hemotoxylin and Eosin stain</p>
 <table style="margin-left:40px;" border="0">
<tr><td colspan="2"  style="font-weight:bold;">a)Harris's hemotoxylin</td></tr>
<tr><td>Hemotoxylin</td><td>2.5g</td></tr>
<tr><td>Absolute alcohol</td><td>25mL</td></tr>
<tr><td>Potassium Alum</td><td>50g</td></tr>
<tr><td>distilled water</td><td>500mL</td></tr>
<tr><td>mercuric oxide</td><td>1.25g OR</td></tr>
<tr><td>Sodium iodate</td><td>0.5g</td></tr>
<tr><td>Glacial acetic acid</td><td>20mL</td></tr>
<tr><td colspan="2"  style="font-weight:bold;">b) Eosin solution</td></tr>
<tr><td colspan="2">Commercially produced Eosin was used.</td></tr>
</table>
<p>All stain dye powders are Gurr dye powders.<br />
  All reagents are analytical reagents, and not Lab reagents</p>
<p><u>Fluorochromes</u></p>
<table border="1" cellspacing="0" cellpadding="0">
  <tr>
    <td><br />
      Manufacturer </td>
    <td>Fluorochrome</td>
    <td>Catalogue Number</td>
  </tr>
  <tr>
    <td>Sigma-Aldrich, Sydney</td>
    <td>Calcein Green</td>
    <td>C0875</td>
  </tr>
  <tr>
    <td>Sigma-Aldrich, Sydney</td>
    <td>Alizarin Complexone </td>
    <td>A3882</td>
  </tr>
  <tr>
    <td>Intervet Australia, Victoria</td>
    <td>Oxytetracycline<br />
      (off the shelf antimicrobial drug)</td>
    <td>Tradename; Engemycin 100</td>
  </tr>
</table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

<p class="jove_content">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<sup>1</sup> and allows for techniques such as fluorochrome assessment and histomorphometry<sup>2</sup>. 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. </p>
<p class="jove_content">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.</p>

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

<p class="jove_content">Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.</p>

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Research

JoVE Journal

Biology

27.0K visualizações.  Monash University. Histologia óssea Undecalcified fornece informações importantes para uma variedade de aplicações clínicas e de pesquisa. É tecnicamente desafiador, especialmente com amostras de grande porte. Este vídeo ilustra o processo de produção de seções de boa qualidade e demonstra as dificuldades técnicas e métodos com os quais a superá-los.