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Scanning Microscopy

Abstract

This study examined the utility of a methacrylate-based embedding medium and microwave staining for light microscopic quantification of hair cells and spiral ganglion cells in the mouse cochlea. The most important phase of the preparation process involved slowing down the polymerization process. The tissue molecules so locked within the plastic matrix produced excellent preservation of the organ of Corti and adjacent structures including the spiral ganglion, as well as tissue ionic charges. Excitable by microwaves, these ionic charges accelerated the movement of the basic dye (hematoxylin) into the tissue, reducing the time for this segment of the staining process from approximately 45 minutes to 1-2 minutes. When embedded in glycol methacrylate (GMA), acidic dyes show less stain-cell affinity so that staining intensity and time cannot be improved significantly. However, addition of color extenders to the counterstain eosin produced distinguished staining of all tissue constituents. Thus, a combination of GMA embedding medium, use of the microwave for staining and addition of color extenders to the counterstain generated excellent structural resolution and contrast. This made both hair cell and spiral ganglion cell counts possible from within the same specimen and provided an opportunity for qualitative evaluation as well.

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