Standard Chemical Fixations as a Basis for Quantitative Investigations of Substances Other Than Deoxyribonucleic Acid

Fixation of living materials in various solutions has been a pillar in the microscopic investigations of higher animals and plants. Most of our basic concepts of cellular and tissue structure rest squarely on these methods of preservation. The original uses for which chemical fixation were intended were entirely morphological and for this purpose they have served faithfully for over one hundred years. The increasing use of histo-chemical methods has made increasingly severe demands on these fixatives. The role of the chemical fixative continues to be an important one, however, because of the ease with which it can be used and because of the insolubility which can be conferred on the particular class of compounds of interest. In the case of quantitative measurements of nucleo-proteins in the cell, a further advantage of chemi-cal fixation lies in the fact that non-wanted substances can be removed (18) so that their unwanted effects on the optical or staining prop-erties of the preparations are eliminated. The fixative of widespread choice for nucleoprotein determinations has been Carnoy’s acetic-alcohol. In an effort to develop a procedure in which proteins of animal nuclei can be quantitatively measured in situ within known limits of varia-bility, the author has been forced to face the old question of whether or not the substances to be measured have been quantitatively retained during the preparatory procedure. This paper is the report of a series of experiments designed to develop a better understanding of the effects of chemical fixation, dehydration, and embedding. METHODS AND MATERIALS Fresh rat tissues were cut into pieces of about 1-2 mm dimensions and several such pieces skew-ered on each of several stainless steel pins. The subsequent handling was done by forceps on the