Staining Myelin Sheaths of Optic Nerve Fibers with Osmium Tetroxide Vapor

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Abstract
OsO4 soln. in water, long regarded as the best fixing and staining agent for myelin sheaths, has poor penetrating power. This peculiarity has limited its use to very small pieces of tissue. The vapor from an aqueous soln. is known to have a much greater penetrating power for non-neural tissues than the soln. itself but nothing has been recorded about its advantages for fixing and staining myelin sheaths of nerve fibers. Difficulties in securing adequate staining of the myelin sheaths in vertebrate optic nerves were overcome largely by the use of the vapor OsO4. The technic is carried out as follows: 1) suspend a portion of the nerve above a 2% soln. of OsO4 for 12-24 hrs. in an air-tight container at room temp.; 2) wash 4-6 hrs. in distilled water, dehydrate in ethyl ale. (50% for 2 hrs., 70% for 2 hrs., and finally 95% overnight), and transfer to n butyl ale. (2 changes of 2 hrs. each); 3) embed in paraffin, section, mount and cover in balsam in the customary manner.

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