Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Summary

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.

Abstract

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 images¹ and allows for techniques such as fluorochrome assessment and histomorphometry². 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.

Protocol

The tissue is placed in a sealed container containing 10% phosphate buffered formalin solution3. Ensure minimal exposure to light, if fluorochrome analysis is to be performed, as the fluorochromes are light sensitive. Establish which orientation of the tissue will be required. 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…

Materials

Solvents:

• 10% Normal Buffered Formalin, Ethanol, Xylene and Butanol acid
  All histology grade, sulfur free.
  Grale Scientific Pty Ltd.
  http://www.grale.com.au

Moulds:

• Peel away, plastic moulds
  Grale Scientific

Slides:

• SUPERFROST PLUS Microscope slides
  Lomb Scientific Pty Ltd.
  http://www.lomb.com.au

Resin:

• GlycolMethacrylate Technovit 7100
  Heraeus Kulzer
  http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm
• Backing Block Resin- Technovit 3040 Histo block
  Heraeus Kulzer

Stains
(Reagents from Grale Scientific)

Goldner’s Trichrome stain

1. Weigert’s iron hemotoxylin (DeGroat)

a) Hemotoxylin solution
Hemotoxylin	1g
Absolute Alcohol	100mL
b) Iron Solution
30% aqueous ferric chloride	4mL
Hydrochloric acid (concentrated)	1mL
distilled Water	95mL

Filtered and added to equal volume of hemotoxylin immediately before stain used.

2. Ponceau-fuchsin-azophloxin stock solutions

a) Ponceau de xylidine solution
Ponceau de xylidine	0.75g
acid fuchsion	0.25g
Acetic Acid	1mL
mix, and add distilled water (100mL)
b) azophloxin solution
Azophloxin	0.5g
Acetic acid	0.6mL
mix, and add distilled water (100mL)
c) Final working stain solution
Ponceau-fuchsin solution	5-10mL
Azophloxin	2mL
0.2% acetic acid solution	88mL

3. Light Green Solution

Light Green	1g
acetic acid	1mL
Mix, and add distilled water (500mL)

4. Phophomolybdic acid-Orange G solution

Phosphomolybdic acid	3g
Orange G	2g
Dissolve in 500mL of distilled water and add crystal thymol.

Von Kossa Stain

1% aqueous silver nitrate

2.5% sodium thiosulfate

1% safranin O

Alcian blue stain

Alcian blue 8GX	1g
3% acetic acid solution	100mL

Hemotoxylin and Eosin stain

a)Harris’s hemotoxylin
Hemotoxylin	2.5g
Absolute alcohol	25mL
Potassium Alum	50g
distilled water	500mL
mercuric oxide	1.25g OR
Sodium iodate	0.5g
Glacial acetic acid	20mL
b) Eosin solution
Commercially produced Eosin was used.

All stain dye powders are Gurr dye powders.
All reagents are analytical reagents, and not Lab reagents

Fluorochromes

Manufacturer	Fluorochrome	Catalogue Number
Sigma-Aldrich, Sydney	Calcein Green	C0875
Sigma-Aldrich, Sydney	Alizarin Complexone	A3882
Intervet Australia, Victoria	Oxytetracycline (off the shelf antimicrobial drug)	Tradename; Engemycin 100