HISTOCHEMICAL DEMONSTRATION OF (Na+-K+)-ACTIVATED ADENOSINE TRIPHOSPHATASE

Rats were perfused with a l2% solution of polyvinyl pyrrolidone (pH 7.25). Frozen sections were prepared from spinal cord and kidney and incubated under conditions that are known to be optimal for demonstrating the p-nitrophenylphosphatase activity of (Na + -K + )-adenosine triphosphatase (ATPase). After incubation, the sections were treated sequentially with cobalt chloride and ammonium sulfide to visualize the reaction product. The reaction product was localized perferentially to the neuropil of the spinal cord and to the distal tubules of the kidney. In addition to the histologic localization, the following observations indicate that the histochemical reaction is specific for demonstrating (Na + -K + )-ATPase activity: (a) the conditions that are optimal for the p-nitrophenylphosphatase activity of purified (Na + -K + )-ATPase were also optimal for our histochemical method; (b) the histochemical reaction was inhibited by ouabain; and (c) the p-nitrophenylphosphatase reaction occurred under conditions that inhibit alkaline phosphatase activity. An additional advantage of this procedure is that it employs a perfusion solution which, although it contains no fixative, preserves the integrity of the tissue after freezing to a degree suitable for light microscopic examination.