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

Abstract The histological study of hard pieces such as tendons, calcified lesions and tissues is an attractive field whose interest is increasing due, among other factors, to the rapid development of implantable prostheses. For these studies, serial sectioning to find the areas of interest throughout the entire piece is very useful, making it possible to specifically apply the appropriate light and electron microscopy techniques in these areas. To achieve these studies, we propose the “Three-Sectioning Method”, which basically consists of subjecting the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest. The pieces were embedded in epoxy resin and cut in thick (approximately 300 µm) sections for the first cycle. The areas of interest were selected on these thick sections and re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1–3 µm). Usually, with these semithin sections, we perform the most relevant techniques for light microscopy and select smaller areas of interest to be cut in ultrathin sections (60–90 nm) for transmission electron microscopy. If it is necessary, we can embed again selected areas of the semithin sections and cut them to obtain new ultrathin sections. All the different kinds of sections we have described here may also be studied under different detector systems of scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnification with a broad variety of histological techniques including electron microscopy.