Preparation of Multi-Purpose Cryosections from Hard Tissue and Large Specimen

Methods: The tooth and the thighbone were dissected from an adult rat after anesthetized and then the specimens were frozen in cooled pentane and freeze-embedded with a water-soluble medium (SCEM). The frozen specimen block was cut with a tungsten carbide blade after placing an adhesive film (Cryofilm) onto the cut surface. The sections were stained histologically and histochemically. Then the section was preserved between the adhesive film and a glass slide with a water-soluble mounting medium (SCMM). The sections were also prepared from a chemically fixed specimen. The section for gene analyses was prepared with a LMD film.

Results: This method produced 4-micron meter thick complete sections from the whole-body mouse, the rat tooth and thighbone without decalcifying. It was possible to make 2-micron-meter thick sections. Each organ was preserved in the whole-body section satisfactorily and cells could be identified clearly. The sections could be used for many kinds of staining such as H-E staining, fluorescence dye staining, the detection of enzymatic activity, and in-situ hybridization. The H-E stained section was prepared within 20 minutes after specimen collection. The sections were used for a fluorescence immunohistochemistry using FITC and DAPI. The fluorescence of GFP-labeled substance was clearly observed. The specimens collected with the LMD film could be used for gene analysis.

Conclusions: The method can produce thin cryosections from undecalcified hard tissues. The sections can be used for staining in a wide range of biological science. These results indicate that this method will become a very important tool in a biological research.