ACTA HISTOCHEM. CYTOCHEM. Vol. 20, No. 1, 1987 LETTER TO THE EDITOR

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1 ACTA HISTOCHEM. CYTOCHEM. Vol. 20, No. 1, 1987 LETTER TO THE EDITOR A SIMPLE METHOD FOR LIGHT MICROSCOPICAL VISUALI- ZATION OF PHOSPHOHYDROLASE ACTIVITIES IN MATERIALS PROCESSED FOR CERIUM- BASED ULTRACYTOCHEMISTRY Since cerium was first introduced into enzyme ultracytochemistry by Briggs et al. (2), it has been extensively used to detect the localization of a wide variety of enzyme activities (1, 6, 10, 12-16). In the case of the phosphohydrolases, cerium captures inorganic phosphates liberated from the substrate and forms cerium phosphate, a fine and consistent electron-dense deposit which surpasses that of lead. Furthermore, using cerium, nonspecific deposits of electron-dense materials so often encountered with lead have seldom been observed (1, 13-15). However, cerium techniques are attended with the fundamental drawback that the reaction products reflecting the localization of enzyme activities at the electron microscopical level are not visible under the light microscope (14, 19). Zimmermann and Halbhuber have described new methods for light microscopical visualization of cerium using lead (4, 5, 19). But besides being complicated, they are not useful at the electron microscopical level. Recently a simple method has been developed in our laboratory for light microscopical visualization of enzyme activities in tissues processed for cerium-based ultracytochemistry. Liver, kidney and small intestine from Wistar rats were used for the demonstration of acid phosphatase (ACPase) and alkaline phosphatase (ALPase), and guinea pig stria vascularis and rat kidney for that of potassium-dependent p- nitrophenyl phosphatase (K-NPPase). Materials were fixed for 1 hr at 0-4 C with 2 % glutaraldehyde in 0.1 M cacodylate buffer containing 5 % sucrose, ph 7.4, for ACPase and ALPase, and with 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M cacodylate buffer containing 5 % sucrose, ph 7.4, for K-NPPase. After washing overnight in 0.1 M cacodylate buffer, ph 7.4, materials were cut into 20 µm thick sections with a DTK-2000 microslicer (Dosaka EM Co., Kyoto). The cytochemical incubation for ACPase and ALPase was done, according to the method of Robinson and Karnovsky (14), for 1 hr at 37 C, and by the method of Kobayashi et al. (10), for 1 hr at 37 C for K-NPPase. After the incubation, sections were washed with 0.1 M Tris-maleate buffer, ph 6.0, for ALPase and K-NPPase, and with 0.1 M acetate buffer, ph 5.0, for ACPase, to remove nonspecific precipitations, and rinsed in distilled water. To detect the reaction products under the light microscope, sections were incubated in 0.1 % lead acetate aqueous solution adjusted with acetic acid to ph 3.9, for 10 min at room temperature, rinsed in distilled water, immersed in 1 % ammonium sulfide for 1 min, and washed briefly with distilled water. Sections of kidney, liver and small intestine were mounted in gelatin jelly and observed. The stria vascularis was embedded in Acrytron E (Mitsubishi Rayon Co. Tokyo), further sectioned 1µm thick and placed on a slide 105

2 106 Okada et al.

3 Light Microscopical Visualization of Cerium 107 glass prior to incubation for light microscopical detection of K-NPPase activity and counterstained with hematoxylin. Figure 1 shows the light microscopical localization of phosphohydrolases. Using the present procedure, sites of positive reaction of enzyme activities are colored brown in all cases. As regards ALPase, the enzyme activity is localized in the brush border of the proximal convoluted tubules of the kidney (Fig, la). Renal corpuscles and distal tubules were not colored brown. In the small intestine, reaction products of ALPase activity were restricted to the striated borders of the absorptive epithelium (Fig. 1B). The positive reaction of ALPase activity is observed as granular structures which are supposed to correspond to lysosomes of various sizes in the liver and kidney. The cortex of the kidney presented intense ACPase activity, especially in the epithelial cells of proximal convoluted tubules (Fig. 1 C). In hepatocytes, granular structures are mainly located around the bile canaliculi (Fig. 1 D, arrowhead 1). Brown coloured granules are also found in Kupffer's cells facing hepatic sinusoids (Fig. l D, arrowhead 2). The K-NPPase activity was studied in the rat kidney and guinea pig stria vascularis. The epithelial cells of the thick ascending limbs of Henle's loops and distal convoluted tubules are stained (Fig, le). Proximal tubules and the thin segments of Henle's loops were devoid of the enzyme activity. The K-NPPase activity in the stria vascularis was confined to the marginal cells (Fig. 1F). Intermediate (Fig. l F, arrowhead 1), and basal cells (Fig. 1 F, arrowhead 2) were not stained. The enzyme activity was not detected in the spiral ligament, Reissner's membrane and epithelial cells covering the spiral prominence. In controls, with substrates omitted or inhibitors added, no positive reaction of these phosphohydrolases was observed in the tissues treated with lead acetate and ammonium sulfide. The present findings of ALPase, ACPase and K-NPPase activities, detected by cerium-based cytochemistry, coincide with those revealed by lead-based techniques (3, 7-9,11,17). The reaction product of phosphohydrolases using lead as the capture agent is lead phosphate. This, in turn, is converted into lead sulfide by ammonium sulfide and can be visualized under the light microscope. Using the analytical electron microscope, the final reaction products of the present method were found to possess both cerium and lead (Fig. 2). This suggests that the reaction deposits of cerium phosphate were not converted into lead phosphate and that either a complex salt or an electrostatic shell of lead around the cerium might have formed. Consequently cerium-lead phosphate can be light microscopically visualized by treatment with ammonium sulfide. The results with lead acetate were compared with those of lead nitrate and lead FIG. 1. Photomicrographs of phosphohydrolase activities. (A) ALPase activity in the brush border of the proximal convoluted tubules of the rat kidney. x 300 (B) ALPase activity in the striated border of the rat small intestine. x 300 (C) ACPase activity is observed as granular structures, which correspond to lysosomes, in the proximal convoluted tubules of the rat kidney. x 600 (D) ACPase activity in (arrowhead 1) hepatocytes and in Kupffer's cells (arrowhead 2), x600 (E) K-NPPase activity is detected in the thick portions of the Henle's loops and in the distal convoluted tubules of the rat kidney. x 300 (F) K-NPPase activity is restricted to the marginal cells of the guinea pig cochlea. The intermediate (arrowhead 1) and basal cells (arrowhead 2) are not stained. x 600

4 Okada 108 et al. FIG. 2. The spot-analysing spectrum of X-ray energy analysis of reaction deposit. lead (PB), phosphor (P) and sulphur (S). Cerium (CE), FIGS. 3A, B. tubules rat kidney. A. Reaction B. Electron products micrographs of ACPase of ACPase activity visualization with lead acetate. After treatment with lead nitrate activity are restricted X 8,000 intense nonspecific in the proximal convoluted of the to lysosomes even after light microscopical precipitation of lead is observed. X 9,000

5 Light Microscopical Visualization of Cerium 109 citrate with regard to the staining pattern of the enzyme activities. Lead citrate, however, is not soluble in the acidic ph range. The localization of enzyme activities were the same with lead nitrate and with lead acetate. With lead nitrate, however, the yellowish background increased. Electron microscopically, nonspecific precipitation of lead was intensely observed in the material treated with lead nitrate (Fig. 3B), but not when lead acetate was employed (Fig. 3A). This could be explained on account of the difference in solubility of these two reagents in water (18). In conclusion, the method reported here is simple, reliable and convenient for the light microscopical visualization of the enzyme activity in material processed for cerium-based ultracytochemistry, and allows the appropriateness of the reaction to be checked before proceeding to electron microscopy. REFERENCES 1. Angermuller, S. and Fahimi, H.: A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Comparison with the lead techniques. Histochemistry 80; , Briggs, R. T., Draht, D. B., Karnovsky, M. L. and Karnovsky, M. J.: Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J. Cell Biol. 67; , Chase, W. H.: The demonstration of alkaline phosphatase activity in frozen-dried mouse gut in the electron microscope. J. Histochem. Cytochem. 11; , Halbhuber; K. J. and Zimmermann, N.: Light microscopical localization of enzymes by means of cerium-based methods. II. A new cerium-lead-technique for alkaline phosphatase. Acta histochem. 77; 67-73, Halbhuber, K. J., Zimmermann, N. and Feuerstein, H.: Light microscopical localization of enzymes by means of cerium-based methods. III. Visualization techniques for cerium phosphate. Acta histochem. 77; , Hardonk, M. J., Kalicharan, D, and Hulstaert, C. E.: Cytochemical demonstration of ATPase activity in the rat kidney basement membrane using the cerium-based method. Acta histochem. Suppl. 31; , Hayashi, M.: Demonstration of acid phosphatase activity using 1-acetyl-3-indolyl phosphate as substrate. J. Histochem. Cytochem. 19; , Hugon, J. and Borgers, M.: Ultrastructural localization of alkaline phosphatase activity in the absorbing cells of the duodenum of mouse. J. Histochem. Cytochem. 14; , Kobayashi, T., Seguchi, H., Okada, T. and Yagyu, K.: Ultracytochemical study of the stria vascularis of the guinea pig cochlea. Anat. Anz. 160; , Kobayashi, T., Okada, T., Seguchi, H. and Tawara, J.: Cerium-based cytochemistry for detection of K+-pNPPase localization. Electron Microscopy 1986, ed. by T. Imura, S. Maruse and T. Suzuki, vol. 3, Biology (1), Japanese Society for Electron Microscopy, Tokyo, 1986, pp Mayahara, H. and Ogawa, K.: Ultracytochemical localization of ouabain-sensitive, potassiumdependent p-nitrophenylphosphatase activity in the rat kidney. Acta histochem. ctochem. 13; , Rechardt, L. and Hervonen, M.: Cytochemical demonstration of adenyl cyclase activity with cerium. Histochemistry 82; , Robinson, J. M. and Karnovsky, M. J.: Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capture agent. J. Histochem. Cytochem. 31; , Robinson, J. M, and Karnovsky, M. J.: Ultrastructural localization of several phosphatases with cerium. J. Histochem. Cytochem , 1983.

6 110 Okada et al. 15. Robinson, J. M.; Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization. J. Histochem. Cytochem. 33; , Veenhuis, M., van Dijken, J. P. and Harder, W.: A new method for the cytochemical demonstration of phosphatase activities in yeasts based on the use of cerous ions. FEMS Microbiol. Lett. 9; , Wachstein, M. and Bradshaw, M.: Histochemical localization of enzymes activity in the kidneys of three mammalian species during their postnatal development. J. Histochem. Cytochem. 13; 44-56, Windholz, M.: The Merck Index, 9th ed., Merck & Co., Inc., Rahway, NJ, , Zimmermann, N., and Halbhuber, K. J.: Light microscopical localization of enzymes of cerium-based methods. I. Detection of acid phosphatase by a new cerium-lead-technique (Ce-Pb-method). Acta histochem. 76; , November 4, 1986 TERUHIKO OKADA, TOSHIHIRO KOBAYASHI, KAZUO HAKOI AND HARUMICHI SEGUCHI Department of Anatomy, Kochi Medical School, Kochi