Silver Protein Staining of Nerve Fibers in Celloidin Sections

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Abstract
Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr. at 37 C, and transferred to 1% concentrated NH4OH in 50% alcohol 15-18 hr. at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 [image] H3BO3/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 g of copper foil, 0.002 in. thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO3, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr. at 37 C, rinsed in distilled water, reduced in a solution of 5% Na2SO3 and 1% hydroquinone for 10 min., washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec., washed in distilled water and placed in 5% Na2S2O3 for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.