Microtechnical Demonstration of Phosphatase in Tissue Sections.

In preliminary experiments the phosphatase activity of aqueous extracts of dog kidney cortex made (a) of fresh ground tissue, (b) of ground tissue dehydrated with several changes of 95% and absolute alcohol, (c) of dehydrated ground tissue exposed to 56°C for one hour was determined by the Berenblum-Chain modification of Kay's method. These experiments have shown that phosphatase is not destroyed either by alcohol or by exposure to 56°C in completely dehydrated state, the maximum decrease in activity observed having amounted to less than 20%. Moreover, purification of phosphatase by alcohol precipitation has been successfully used by Mart-land and Robison. It appeared possible to demonstrate phosphatase in celloidin or paraffin sections on the basis of the following principle: If tissue sections containing active phosphatase are incubated with a solution of sodium glycerophosphate or of some other suitable ester-phosphate, such as hexose-phosphate or nucleinate, at a suitable pH, at the sites where phosphatase is present, PO4 ions will be split off. These ions may be trapped at the spot by the salts of metals whose phosphates are insoluble. A precipitate of insoluble phosphate forms which, if visible, or made visible, indicates the presence of phosphatase. A useable technic has been worked out on the basis of this principle.Fixation. Phosphatase is dissolved or destroyed by most of the routine fixatives. In alcohol, however, it is preserved for many weeks at least. Thin slices of fresh tissue should be fixed in 95% alcohol for about one day.Unfortunately, decalcification is impossible, because all acids destroy phosphatase completely.Embedding. Both celloidin and paraffin embedding is suitable. If the tissue is well dehydrated, exposure to paraffin at 56 to 60°C for 2 hours will do no harm.