Faculty of Pharmacy, University of Manitoba, Apotex Center, 750 McDermot Avenue, Winnipeg, Manitoba, Canada.
J Neurosci Methods. 2010 Mar 15;187(1):59-66. doi: 10.1016/j.jneumeth.2009.12.013. Epub 2009 Dec 28.
Histological analysis of bone encased tissue is severely hampered by technical difficulties associated with sectioning calcified tissue. Cryosectioning of bone is possible but requires significant technical adaptation and expensive materials and is often time-consuming. Some decalcifying reagents in common use result in successful cryosectioning in less time but the integrity of the soft tissue of the spinal column is often compromised during processing. This can result in significant loss of cellular detail. In order to find a method that would allow cryosectioning of the bone without loss of structural integrity of the underlying soft tissue we assessed the efficacy of four different decalcifying reagents with respect to their effects on the cellular structure of the myelin of the grey and white matter of the spinal cord. The antigenic integrity of the spinal cord white matter was evaluated using tissue structural integrity and quality of myelin basic protein immunostaining. The result of this research shows that 6% TCA not only decalcifies intact spinal column suitably for cryosectioning but does so without compromising the antigenic integrity of the tissue. The ease of application, speed of processing and a favorable cost-effective profile were secondary benefits noted with the use of the 6% TCA decalcifying solution. The ability to utilize a decalcifying solution that allows for both histomorphometry and immunohistochemistry in the same spinal column segment represents a novel technique that will provide new insights into pathophysiological aspects and therapeutic approaches ispinal cord damage or disease.
对包裹在骨组织中的组织进行组织学分析，受到与钙化组织切片相关的技术困难的严重阻碍。骨的冷冻切片是可能的，但需要进行重大的技术调整、使用昂贵的材料，并且通常很耗时。一些常用的脱钙剂可以在更短的时间内成功地进行冷冻切片，但在处理过程中，脊柱的软组织完整性往往会受到损害。这可能导致大量细胞细节的丢失。为了找到一种方法，可以在不损失骨下软组织结构完整性的情况下进行冷冻切片，我们评估了四种不同脱钙剂对脊髓灰质和白质髓磷脂细胞结构的影响。使用组织结构完整性和髓磷脂碱性蛋白免疫染色评估脊髓白质的抗原完整性。这项研究的结果表明，6%TCA 不仅可以适当地对完整的脊柱进行脱钙，以进行冷冻切片，而且不会损害组织的抗原完整性。使用 6%TCA 脱钙剂还具有易于应用、处理速度快和具有良好的成本效益等次要优点。能够在同一脊柱段同时进行脱钙、组织形态计量学和免疫组织化学分析的能力代表了一种新技术，它将为脊髓损伤或疾病的病理生理和治疗方法提供新的见解。
