脱钙骨组织学，形态计量学和荧光染料分析制备
Summary
脱钙骨组织学各种临床和研究中的应用提供了重要信息。这在技术上是具有挑战性，特别是大尺寸的标本。该视频演示了质量好的部分生产过程和演示的技术难点和方法，克服这些。
Abstract
脱钙骨组织学显示骨微架构。它同时显示矿化骨，它提供了对骨代谢或骨形成和骨吸收的重要信息和细胞成分。这具有巨大的重要性在各种不同的临床和研究中的应用。它会产生美丽的图像技术，如荧光评估和组织形态计量学2 1和允许。荧光分析是一种钙结合的荧光染料注射在特定的时间点，在那个特定的时间允许的矿化量的量化技术。组织形态计量学是在微观层面的骨定量过程。
执行脱钙骨组织学是在技术上具有挑战性，特别是大尺寸标本。它要求在技术标准石蜡嵌入组织学的变化。该视频演示了质量好的部分生产过程和演示的技术难点和方法，克服这些。实现方式类似于其他软组织的密度和骨的通透性较低，需要相当长的固定和处理时间，但是由于经常服用几个星期，试样制备，固定和处理。嵌入使用类似或等于硬度和密度，如甲基丙烯酸甲酯树脂骨支撑介质，但不像石蜡渗透和嵌入，这是一个不可逆转的步骤。切片可以通过研磨产生较厚的部分，这是最适用于如荧光分析的研究。这是最好的实现上macrotome使用的钻石刀片。另外，较薄，可产生光镜，这是一个非常锋利的刀片使用大锤切片。爬犁切片提供了额外的的大，硬模块所需的强度和稳定性。树脂包埋切片可染各种各样的污渍，这是证明。
Protocol
Acknowledgements
作者要感谢她的专长苏康奈尔女士的协助下，在树脂嵌入和她的实验室技术女士千金墓群。作者要感谢他们的意见弗兰克Kandziora教授和博士玛丽 – 安妮Polboth。
Materials
- 10% Normal Buffered Formalin, Ethanol, Xylene and Butanol acid
All histology grade, sulfur free.
Grale Scientific Pty Ltd.
http://www.grale.com.au
- Peel away, plastic moulds
Grale Scientific
- SUPERFROST PLUS Microscope slides
Lomb Scientific Pty Ltd.
http://www.lomb.com.au
- 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
(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 |
Referências
- Kerr, J. . Atlas of Functional Histology. , 164-168 (2000).
- Parfitt, A. M. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 2, 595-610 (1987).
- An, Y. H., Martin, K. l. . Handbook of histology methods for bone and cartilage. , (2003).
- Bancroft, J. D., Gamble, M. . Theory and Practice of Histological Techniques. , 352-360 (2008).
