Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination

The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method saves time and material and provides equivalent ultrastructural information. Furthermore, thin sections from untreated material are significantly larger and more homogeneous, more resistant to the electron ray, as well as more suitable for sectioning. The electron microscopical pictures obtained allow a comparison to previous test results achieved with fixed and embedded material. Using the preparation of thin sections from untreated material method, fixation and embedding artifacts are avoided, providing a clearer interpretation of the electron microscopical findings. Considerable advantages are achieved by using immunohistochemical techniques with untreated horn specimens because fixation invariably decreases antigenicity.