Axonal microtubules and associated filaments stained by alcian blue

Delicate filamentous projections 3–8 nm in thickness radiate laterally from the surface of microtubules in crayfish ventral axons and anastomose with similar filaments projecting from neighbouring microtubules and with unattached filaments in the axoplasmic matrix. Following Alcian blue treatment, a layer of alcianophilic material 5–30 nm thick was revealed on the axoplasmic surface of all intra-axonal structures, including microtubules, and the filamentous projections possessed significantly enhanced contrast. In Alcian blue-penetrated axons these prominent filamentous projections were organized into a conspicuous axoplasmic network connecting microtubules up to 350 nm apart. Intra-axonal alcianophilia was nearly abolished when connectives were stained in the presence of 0·1–0·2 M MgCl2 or when pretreated with hyaluronidase. The filamentous elements and the exterior surface of microtubules retained some degree of alcianophilia when stained at pH 5·5 and 4·1 but few deposits were associated with intra-axonal membrane systems. These observations are consistent with the hypothesis that the projecting filamentous elements and perhaps the exterior surface of the microtubule walls have, at least in part, a carboxylated polyanionic character.