Staining Paraffin Sections with Protargol 6. Impregnation and Differentiation of Nerve Fibers in Adrenal Glands of Mammals

A series of experiments with protargol staining of nerve fibers in mammalian adrenal glands has yielded the following procedure: Fix-1-2 days in a mixture of formamide (Eastman Kodak Company) 10 cc, chloral hydrate 5 g., and 50% ethyl alcohol 90 cc. Wash, dehydrate and embed in paraffin. Cut sections about 15 and mount on slides. Remove the paraffin and run down to distilled water. Mordant 1-2 days in a 1% aqueous solution of thallous (or lead) nitrate at 56-60°C. Wash thru several changes of distilled water and impregnate in 1% aqueous protargol (Winthrop Chemical Company) at 37-40°C. for 1 to 2 days. Rinse quickly in distilled water and differentiate 7-15 seconds in a 0.1% aqueous solution of oxalic acid. Rinse thru several changes of distilled water for a total time of 0.5 to 1.0 rain. Reduce 3-5 rain, in Bodian's reducer: hydroquinone 1 g., sodium sulfite 5 g., distilled water 100 cc. Wash in running water 3-5 min. and tone 5-10 min. in a 0.2% gold chloride solution. Wash 0.5 min. or more and reduce in a 2% oxalic acid solution to which has been added strong formalin, 1 cc. per 100. (Caution. This last reduction is critical and over-reduction can spoil an otherwise good stain; 15-30 seconds usually suffices, and the sections should show only the beginning of darkening to a purplish or gray color.) Wash, fix in hypo, wash, dehydrate and cover.