RH 414 a New Dye to stain non - Decalcified Bone Tissue

Histological analysis of hard tissue is hampered by the fact that embedding in methacrylate reduces the spectrum for staining and labelling using monoclonal antibodies. It was the aim of this study to develop a new technique of sample preparation and histological analysis using confocal laser scanning microscopy (CLSM).Bone samples from the medial cortex of proximal tibiae of 3 dogs were removed during implant surgery and fixed in 4% formaldehyde for histological analysis. The material was divided into 4 pieces of similar size, the formaldeyde was removed, subsequently the specimens were stained for 24 h using RH 414 or DiOC 6/7 (Molecular Probes), which have been used for membrane labelling. The stained bone samples were analyzed in the Leica TCS 4D confocal laser scanning microscope (CLSM). Then they were embedded in paraffin or in laromin C 268 (BASF, Ludwigshafen) and thinned by grinding (Donath, 1982) for parallel analysis using CLSM, light (LM) and fluorescence microscopy (FM).Bone cells with their typical morphology could easily be identified by LM and CLSM, but not by FM. Cellular elements were still visible even when the focal plane was adjusted 60 µm below the sample surface. Optical sections in x/z-direction revealed cells even 100 µm below the surface irrespective of the mode of sample preparation. The newly developed technique allows microscopic analysis of cellular elements on the surface and below the surface without decalcification of the bone sample.