Pyridine-silver methods for the study of optic axons in retinal whole mounts

The optic fibers in the retinas of diverse species may be selectively stained and viewed en bloc in the embryonic and adult states. Treat the eye as follows: 1) 50% pyridine for at least 16 hr, 2) distilled water 3--4 hr, 3) 20% H2O2 until the eye is a light brown, 4) 95% ethanol overnight, 5) 1.5% AgNO3 for 2--6 days at 37 C, 6) in water, remove the vitreous, then direct 0.25% pyrogallic acid in 1.25% formalin against the retina for 2--5 secs until the optic fibers are reduced to a coffee-copper color (1--4 minutes), 7) dissect the retina and mount flat on a glass slide, 8) cover with glycerin, apply a coverslip, and fix in place with nail polish. Variants for particular species are given. The technique offers an advantage over Golgi and methylene blue methods which tend to stain only a small percentage of fibers and frequently do not work at the earliest stages of development.