Ultrasonic microdissection of rat cerebellum for scanning electron microscopy

The cerebelli of rats were initially fixed with aldehydes (modified Karnovsky's fixative; 503 mOsM/L) by cardiac perfusion. Blocks of tissue were razor-cut, usually longitudinal to folia, and immersed in the same fluid for 2-4 hours. Three separate methods of treatment followed: (1) immersion in 1% aqueous boric acid, or (2) in 2% phosphate buffered OsO4 followed by boric acid or (3) in an 8/2 mixture of boric acid and OsO4. After 18-48 hours immersion the blocks were dehydrated in ascending grades of acetone. They were then exposed to ultrasound in 100% acetone at frequencies of 80 kHz or 40 kHz for 10 to 20 minutes. Microdissection of cut surfaces (erosion) occurs after all three treatments. It is least extensive after boric acid, moderate after OsO4 and greatest after the combined mixture. All cerebellar cell types are recognizable as are numerous fibers according to morphology and position. Variable erosion accommodates analysis of different levels of neural organization. In general, structural situations not involving great depth of field are best revealed by H3BO3 or OsO4. Blood vascular relationships to other structures are best demonstrated in deeply eroded specimens.