Preparation of Thin Undecalcified Frozen Sections from Hard Tissue

Objectives: Thin nonfixed and undecalcified sections from bone and tooth are required for histochemical dental studies because fixation and decalcification during specimen preparation cause protein denaturation and loss of water-soluble materials. The purpose of this study is to prepare undecalcified sections from an extracted adult human tooth, a rat alveolar bone with tooth, and a rat thighbone.

Methods: A 7-month-old rat was given a 0.1% calcein solution intraperitoneally, anesthetized after 3hours, and then the alveolar bone and the thighbone were dissected. The specimens were frozen in pentane cooled with liquid nitrogen and freeze-embedded with an embedding medium (SCEM). Human tooth specimen was embedded using the same procedure. The frozen specimen block was fixed to the cryomicrotome stage and trimmed with a tungsten carbide disposable blade. The block surface was then covered with a pressure sensitive adhesive film (Cryofilm) and cut into 2-6 micron meter thick sections with a cryomicrotome (Leica CM3050S). The sections were histologically or histochemically stained, and then permanently preserved between the adhesive film and a glass slide with photopolymerization resin (SCMM-R2).

Results: This technique yielded 4-micron meter thick complete sections from the adult human tooth as well as the alveolar bone and tooth of a rat. It was possible to make 2-micron meter thick sections. Both soft and hard tissues were well preserved. Cell characteristics (osteoblasts, odontoblasts and fibroblasts) can be observed by using a light microscope at high power. Enzymatic activity and immune-reactivity were stronger than those of chemically fixed specimens. Calcein fluorescence was clearly observed on the bone surface of the 2-micron meter thick sections.

Conclusions: This section preparation method is very useful for preparing undecalcified sections. These sections can be used for the histological study, histochemical study, enzyme histochemistry, and immunohistochemistry. Other applications are possible.