Block Intensification of Neurones Stained with Cobalt Sulphide: A Method for Destaining and Enhanced Contrast

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Abstract
In characterizing neurones it is important to have both physiological and morphological information. This was first realized in a practical sense by Stretton & Kravitz (1968), who explored the use of the Procion series of dyes and used them to study the morphology of identified lobster neurones. Initially, Procion dyes were injected into single neurones using microelectrodes. However, it was later found that Procion Yellow could be introduced into the cut end of axons (Iles & Mulloney, 1971). This made it possible both to stain small neurones which would be hard to impale with microelectrodes, and also to determine rapidly the position of the cell bodies of neurones with axons in a given nerve trunk. Subsequently, it was found that neurones could be stained by introduction of cobalt ions followed by the formation of a black precipitate of cobalt sulphide (Pitman, Tweedle & Cohen, 1972). This method had the advantage that intact neurones could be observed in whole-mount preparations. A great improvement on the resolution of the cobalt sulphide method was obtained using a modification of the Timm ‘s sulphide-silver intensification procedure (Tyrer & Bell, 1974). This made it possible to visualize fine branches of neurones which were too lightly stained to be seen using the original cobalt sulphide method. It is also relatively permanent, whereas unintensified preparations often fade. However, a disadvantage of this modification was that it required sectioning of the tissue before intensification. More recently, block intensification methods have been described (Strausfeld & Obermayer, 1976; Bacon & Altman, 1977). These permit whole-mount viewing of entire neurones with intensely stained fine processes.