A Comparison of Fixation by Formaldehyde and Glutaraldehyde-Formaldehyde for Combined Light and Electron Microscopy of Axonal Degeneration in the Mammillary Body

Mammillary body (Mb) cell structures were compared following four different procedures for fixation, in order to find one procedure which would be suitable both for silver impregnation methods and electron microscopy. Fixatives compared were: (1) formaldehyde (4% pH 5.5), Mb from a frozen-section; (2) formaldehyde (4% pH 7.2.), Mb from a frozen-section; (3) glutaraldehyde-formaldehyde (1%-4% pH 6.0), Mb from a frozen-section; and (4) glutaraldehyde-formaldehyde (3%-2% pH 7.2), unfrozen Mb. All solutions were perfused into the left ventricle of the heart of anesthetized rats, except for the animal from which the unfrozen tissue was obtained. The Mb was excised from the fresh brain and immediately placed in a fixative solution. After aldehyde fixation, the tissues were further fixed in 1% OsO₄ (pH 7.4), dehydrated, embedded in Epon-Araldite and double-stained with uranyl acetate and lead citrate. Comparisons of mitochondria, myelin, synapses, neurofilaments and synaptic membranes indicated that the perfusion of 4% formaldehyde is suitable for both electron microscopy and silver impregnation, and was preferable to the other fixatives tested.