A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

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Abstract
Extensive exper. with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections 15-25 microns, frozen sections 25 to 40 microns. Place sections for 24 hrs. in 50% alc. to which 1% by vol. of NH40H has been added. Transfer the sections directly into a 1% aq. soln. of protargol, containing 0.2 to 0.3 g. of electrolytic Cu foil which has been coated with a 0.5% soln. of celloidin, and allow to stand for 6 to 8 hrs. at 37[degree] C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24-48 hrs. at 37[degree] C. directly into a pyridinated soln. of alcoholic protargol (1.0% aq. soln. protargol, 50 ml.; 95% alc, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated Cu. Rinse briefly in 50% alc. and reduce 10 min. in an alkaline hydro-quinone reducer (H3B03, 1.4 g.; Na2SO3, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroughly in water and tone for 10 min. in 0.2% aq. AuCl3, acidified with acetic acid. Wash in distilled water and reduce for 1-3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3-5 min. with 5% aq. Na2S2O3 + 5H2O. Wash in water and stain overnight in Einarson''s gallocyanin. Wash thoroughly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock soln., 20 ml.; distilled water, 30 ml.) of the following stock staining soln.: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid. 8 ml. and stain for 1 hr. Differentiate with 70% and 95% alc; pass the sections through butyl alc. and cedar oil; mount.