Enzyme Digestion of Epon Embedded Akinetic Cilia
Terminal gill cilia of the freshwater mussel Unio stop beating when exposed to hyperosmolar solutions. Ultrastructural studies of the cilia during akinesia showed the absence of the 9+2 doublet microtubules and the ciliary matrix was amorphous and extremely electron dense (Fig. 2). It seemed possible that the axonemal microtubules might not be disrupted by shock-stopping cilia, but rather were obscured by the formation of the pleomorphic matrices. This hypothesis was tested using a modified enzymatic proceedure which has been shown to specifically digest spermatozoa flagellar microtubules in Epon ultrathin sections.Terminal gill filaments with control cilia beating in aged tap water and akinetic cilia which had been exposed to 0.154 M NaCl were fixed at 22°C for 30 min in 3% phosphate buffered (pH 7.35) glutaraldehyde and post-fixed at 22°C for 30 min in 2% buffered osmium tetroxide. The gills were rinsed in buffer, dehydrated in ethanol and embedded in Epon 812.