SELECTIVE AXONAL ARGENTAFFIN STAINING IN RAT CENTRAL-NERVOUS-SYSTEM AFTER PROTEIN MERCURATION

The mercury-silver (Hg-Ag) argentaffin technique, known to stain speci fically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats . Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon an d dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, fre shly prepared by adding concentrated ammonia to 60% (w/v) silver nitra te solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) s odium sulfite and distilled water. All steps were carried out using da rk-colored glass flasks. Samples were dehydrated with ethanol and embe dded in Paraplast or Poly Bed, Electron microscopy showed the silver-r educing protein inside the axons. Methylation abolished Hg-Ag axonal r eactivity indicating that carboxyl groups were necessary for silver st aining. Proteins with solubility properties characteristic of neurofil ament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, whi le basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differen ces in cytoskeletal neurofilaments.