Silver Staining, Featuring Rapid Reduction, For Whole Mounts of Retina and Optic Pathways in Chick Embryos

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5-7 hr with hourly changes of distilled water or with running tap water for 4-6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16-48 hr. (4) Impregnate with 1.5% AgNO₃ for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2-5 sec. Wash the eye with distilled water 30-60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20-30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.