Histochemical Localization of Alkaline Phosphatase in The

CHEMICAL investigations have shown that alkaline phosphatase is present in the retina (De Conciliis, 1934; Reis, 1951). In these investigations the whole retina was homogenized and the enzyme estimated in extract. Obviously the estimations of enzyme activity by such a necessarily gross method give no idea of the exact distribution of the enzyme in this, functionally, so finely differentiated tissue. This object may only be achieved by using histochemical methods. A knowledge of the histological localization of the phosphatase in the retina, may perhaps enable us to make some suggestions about its physio logical role there. Gomori (1939) described a method of demonstrating alkaline phosphatase activity in tissue slides. This method consists in incubating the fixed tissue slides with buffered solution of a phosphate ester and a calcium salt. The phosphatase hydrolyses the ester, and the liberated phosphate forms a precipitate of calcium phosphate. The latter is converted to cobalt phosphate (by the addition of cobalt nitrate solution) and this with ammonium sulphide produces black, insoluble, cobalt sulphide. Thus black deposits of cobalt sulphide are formed at the sites of phosphatase activity. This technique was used by Siillmann and Payot (1949) in the study of corneal epithelium phosphatase. Fran9ois and Rabaey (1951), and simultaneously De Vincentiis (1951), published their investigations on the phosphatase activity of different ocular tissues. Since in the described method phosphatase activity is visualized as a brown or black deposit, it is difficult to differentiate it from the similarly coloured melanin pigment. Francois and Rabaey conunent on this difficulty in their paper. They make the final observations by comparing the phosphatase-stained slides with control slides. If melanin is present in the control, the activity of phosphatase is demonstrated when a darker or more abundant staining seems apparent in the incubated slide. De Vincentiis (1951) found another solution to this problem. For the study of phosphatase in uveal membranes he used albino rabbits-thus avoiding the presence of mel.nin, but limiting his research to these animals only. The disadvantage of the method used in these papers is that calcium phosphate is not wholly insoluble but is apt to be diffused to a certain extent. As a result, deposits may occur which are not a true indication of phosphatase activity. There also exists a tissue affinity for cobalt salts, so that the cobalt