Fixation of Soft Tissue Surrounded by Bone with Microwave Irradiation: Electron Microscopic Observation of Guinea Pig Inner Ear

Abstract

When electron microscopy is performed on organs such as the inner ear that cannot be removed immediately after decapitation of animals, it is necessary to fix the target organ or tissue by systemic or regional perfusion fixation. However, such methods of fixation can increase vascular pressure or perilymphatic pressure, making it difficult to perform precise morphological observation of the vascular endothelial cells and membranous labyrinth. We recently attempted fixation of the cochlea by microwave irradiation. Guinea pigs were decapitated. The bullas were then removed from each animal and fixed in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde. Microwave (300 W) irradiation was then applied to the specimen for 1 minute. The fixative was immediately replaced with new fixative (4°C). This sequence of manipulations was repeated 10 times, for a cumulative microwave irradiation time of 10 minutes. During the microwave irradiation period, the fixative temperature was kept at about 30°C. After the last round of irradiation, the specimens were kept immersed in the fixative for 1 hour. After a small slit was created in the bone on the lateral wall of the cochlea, the specimens were post-fixed in osmic acid and embedded in Epon 812. Each specimen was cut into halves along the plane containing the modiolus of the cochlea. After the bone on the lateral wall of the cochlea was cut off under a stereoscopic microscope, ultrathin sections were prepared for observation under a transmission electron microscope. With this technique, the stria vascularis and the organ of Corti were fixed to a degree comparable to or better than that achieved with the conventional method of fixation. Fixation with microwave irradiation is relatively simple and can solve the problems associated with perfusion fixation, and thus provides an excellent means of fixation. This technique appears to be particularly promising for fixation for soft tissue surrounded by bone.

Keywords

electron microscope inner ear morphology microwave irradiation

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