Histochemical demonstration of succinic dehydrogenase activity in tissue sections by a modified technique.

Abstract

A method for the histochemical demonstration of succinic dehydrogenase was first described by Seigman and Rutenburg in 1951 (7). Their technique utilized ditetrazolium chloride (BT) as an indicator dye, and the detection of reduced BT as diformazan constituted a positive test for succinic dehydrogenase activity. This initial report has been followed by two subsequent papers describing the distribution of succinic dehydrogenase in various tissues and organs of several mammals (5, 7). Two modifications of the original method have been employed in these later studies; the use of Al , Ca , and HCO3activating ions and of anaerobic conditions (nitrogen bubbling, etc.) have been found to be beneficial and more critical in demonstrating sites of low succinic dehydrogenase activity. Of further interest is the paper of Shelton and Schneider showing that among several of the tetrazoles used, neotetrazolium was the most efficacious as an indicator of dehydrogenase activity (9). The latter is in accord with the findings of Padykula (5), Malone (4) and the present workers. As a result of the difference of opinion regarding the choice of the most critical indicator dye and the consequent lack of agreement in the qualitative detection of succinic dehydrogenase activity in a number of tissues and organs previously described as having questionable or a low degree of activity, the present study was undertaken in an effort to: (1) critically evaluate the existing differences regarding this technique, and, (2) outline a method which is simple, direct, and efficient in demonstrating succinic dehydrogenase activity in many tissues preP viously described as negative for this test. Various tissues were removed from albino rats and frozen immediately on a block of dry ice. Tissues in this frozen state may be stored for long periods of time without any noticeable loss of enzyme activity. In preparation for the sectioning procedure, the tissues were trimmed and mounted by freezing to the stage of a carbon dioxide freezing microtome. Fresh frozen sections were cut at 10 to 20 and mounted upon slides by drying in air at room temperature for a short period of time (5 to 10 minutes). Pending the sectioning of other tissues, the mounted sections were stored at 4#{176}C. in 0.1 M sodium phosphate buffer contain-