A novel decalcification method for adult rodent bone for histological analysis of peripheral-central nervous system connections

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. (C) 2009 Elsevier B.V. All rights reserved.