Three-sectioning method: A procedure for studying hard tissues and large pieces under light and electron microscopy.

The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 μm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 μm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.