Demonstration of phosphorylase activity in the rat brain

Summary The phosphorylase reaction in rat brain is strong, but for its correct evaluation in pathological states certain precautions are needed. Control material from animals of the same age should be used identically and simultaneously with the pathological, preferably freezing blocks from each on the same holder. Several sections should be cut and then the holder rotated through 180° and more sections cut, thus avoiding changes in section thickness which can drastically influence the color reactions, i.e., apparent phosphorylase activity. The time the animal is killed until its brain is frozen should be less than 5 min, preferably 11/2 to 3 min, to limit postmortem changes in the enzyme.Preparing the stock incubating medium and storing it at 4°C does not affect the potency of the medium; this procedure is convenient and eliminates more variables.Small amounts of medium can be placed between two slides set at right angles to each other. The height of the chamber, equal to the thickness of a slide, is uniform.Prefixation of the cryostat sections in cold acetone (4°C) for 5 min can be useful for obtaining sharper enzyme localization.AMP is the nucleotide of choice for stimulating full phosphorylase reaction. The inclusion of glycogen in the incubating medium is also necessary.The optimal incubation time for phosphorylase + branching enzyme is about 1/2 hr for 16 sections. For phosphorylase alone (active or total) it is about 2 hr. Dehydrating, clearing, and mounting in iodine-containing solutions is not recommended nor is it necessary. The best preservation of original colors, which is important for the correct interpretation of the results, is accomplished by sealing the preparations mounted in iodine-glycerin with paraffin.