M. Wachstein, E. Meisel
Nov 1, 1956
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Influential Citations
395
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Journal
Journal of Histochemistry and Cytochemistry
Abstract
Chiquoine (J. Histochem. Cytochem. I, 429, 1953, and III, 471, 1955) developed a technique for the histochemical demonstration of glucose-6-phosphatase, one of the most important enzymes in intermediary carbohydrate metabolism. He converted the barium salt of glucose-6-phosphate into the Potassium Salt and incubated fresh frozen sections fixed to slides in a mixture which contained the glucose-6-phosphate and lead nitrate adjusted to a pH of 6.7. With some modifications of this technique, we have obtained consistent and reproducible results. Poor staining in tissues which contain large amounts of this enzyme, as for instance liver, as has been described by Chiquoine, was not encountered. We used the following procedure: Free-floating fresh frozen sections cut at a thickness of 10-15 s with a Sartorius freezing microtome are brought into a substrate mixture consisting of 20cc of a 125 mg% solution of potassium glucose-6-phosphate (obtained from Sigma Chemical Company, St. Louis, Missouri), 20cc of a 0.2M tris-(hydroxymethyl)aminomethane maleate (tris-Maleate) buffer pH 6.7, 3cc of a 2% lead nitrate solution and 7cc of distilled water. Following incubation for 5-15 minutes at 32#{176}C the sections are washed, treated in the usual manner with dilute ammonium sulfide, washed again and postfixed with a 6% solution of neutral formalin. They are then covered with glycerin and the coverslips rimmed with nail-polish. With the use of the now available potassium salt of glucose-6-phosphate, enzymatic activity in the rat kidney is almost exclusively limited to the proximal portions of the proximal convoluted tubules with only slight staining of their straight terminal portions. When the barium salt is used, however, the terminal portions of the convoluted tubules stain stronger and in addition ascending limbs of Henle’s loops as well as capillaries in the glomeruli give a moderately strong staining reaction. A varying but occasionally not inconsiderable amount of activity is observed in control sections treated in an identical manner with the exception that in the substrate mixture glucose-6-phosphate is replaced by glycerophosphate. This is particularly noticeable in sections incubated for more than 10 minutes. M. WAcH5TEIN and E. MEISEL Department of Pathology St. Catherine’s Hospital Brooklyn, N. Y.