This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

An account is given of an investigation of histochemical methods for calcium using standard test tissues (McGee-Russell, 1957) known to contain calcium salts, as test objects upon which to assess the success, reproducibility, and mutual consistency of methods. The methods for the revelation of calcium in the literature fall into two groups: metal substitution techniques, and dye-lake reactions. Published methods have been tested and many have been found of little use for cytological investigations. A set of six metal substitution techniques, including a new copper dithioxamide method, has been established as giving useful results. The empirical basis of the staining reactions for calcium with dyestuffs has been investigated, and sensitive and specific techniques using anthraquinone derivatives are outlined. The significance of pH in dye-lake reactions is emphasized, and it is pointed out that previous results with anthraquinone dyestuffs have been achieved by an empirical adjustment of pH in the staining solution. The necessity of making a distinction between the formation of a dye-lake precipitate as a true histochemical positive, and staining effects which may occur upon tissue components in the neighborhood of calcium deposits, through the interaction of the dye, the dye-lake, the calcium salt, and the matrix, is stressed. An oxazin dyestuff, gallamine blue, recently introduced for the staining of calcium deposits, was found to be inconsistent in its results, and capable of giving misleading information. Its general use is not recommended. The possibility of studying calcium deposits by means of the presence of contaminating metals is considered. The reagent sodium rhodizonate was found to give very fine localization of known calcium deposits in a number of tissues.

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