An Improved Histochemical Technic for Acid Phosphatase

Causes of failure in the histochemical technic for acid phosphatase were studied. It was found that the ratio of buffer to substrate in the incubating mixture is an important factor in the constancy of results. In the case of glycerophosphate as a substrate, the optimal ratio is 5 to 6; with other substrates it may be different. The mechanism of the effect of this ratio on the results is explained.     

Histochemical Localization OF Acid Phosphatase IN Germinating Maize Kernels

Sections of germinating maize kernels obtained by freezing technic were examined for acid phosphatase according to the histochemical method of Gomori. The embryonic axis, scutellum and aleurone layer were found positive for the enzyme. A microtechnical method for preformed phosphate, based on Sumner's colorimetric method for phosphorus, is described.     

Demonstration of Various Acid Hydrolases and Preliminary Characterization of Acid Phosphatase in Naegleria fowleri1

ABSTRACT. Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include β-glucosidase, β-galactosidase, β-fucosidase, α-mannosidase, hexosaminidase, arylsulfatase A, and β-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly “acid” hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: 1) pH optima, 5.5; 2) K_m (4-methylumbelliferyl phosphate), 0.60 mM; 3) molecular weight (estimated by gel filtration chromatography), 92,000; 4) inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

Histochemical Demonstration of Pyrophosphatase

Fresh tissue slices were fixed in 5% formalin containing 0.9% NaCl for 10-20 min and frozen sections therefrom floated for 3 hr at 37°C on an incubating mixture made as follows. Sodium pyrophosphate (Na₄P₂O₇-12H₂O), 1.088 gm was dissolved in 20-30 ml of distilled water and to this was added ferric chloride (FeCl₃-6H₂O), 0.61 gm dissolved in 10-15 ml of water. The precipitate was just dissolved by cautiously adding 5-10% aqueous Na₂CO₃ solution and the pH adjusted to 7.2 with 1N HCl. The volume was made up to 100 ml and 0.9 gm of NaCl added. Before use, 1 ml of 10% Mg(NO₃) was added. After incubation, sections were washed 10-15 min in 0.9% NaCl, then mounted on glass slides and air-dried. When dry, the slides were immersed in 0.9% NaCl containing 0.2-0.5% ammonium sulfide for 2-3 min, then dehydrated rapidly through graded alcohols, cleared, and covered in balsam. Sites of pyrophosphatase activity stained in various shades of green. Acid pyrophosphatase also was histochemically demonstrated by the same principle, excepting that the substrate solution was adjusted to pH 3.7-4.0 with acetate buffer. The pattern of distribution of pyrophosphatase and glycerophosphatase was almost identical.     

Isoenzymes of Acid Phosphatase and Non-specific Esterases in Cultures of Neoplastic and Normal Tobacco Tissues

Axenic cultures of normal, habituated and crown gall teratoma tissues were grown under varying conditions to examine the effects of environment on the expression of neoplastic character. Acid phosphatase patterns on polyacrylamide gels did not vary greatly among tissues although there were differences in acid phosphatase activity between various strains of Agrobacterium tumefaciens , the bacteria which cause crown gall. Certain esterase isoenzymes were found only in tissues grown on specific media, while others were tissue-specific but independent of the nature of the medium. Comparisons of liquid and solid grown cultures revealed that culture conditions also influence esterase expression. Both sunflower and tobacco crown gall tissue contained an esterase not found in habituated or normal tissues, and similar in electrophoretic mobility to an esterase found in extracts of the bacteria that had induced the tumors. The basic difference between the three tissue types studied is the manner in which they respond to a given environment.     

Improvement in the Histochemical Demonstration of Acid Phosphatase, β-Galactosidase and Nonspecific Esterase in Glycol Methacrylate Tissue Sections by Cold Temperature Embedding

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, β-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.     

The Histochemical Detection of iron in Tissues

The fixation and staining of iron in tissues is discussed. Procedures for demonstrating iron in hemoglobin and nuclei are also briefly considered.

Lillie's formalin buffered at neutrality gave the optimal fixation for iron. The Prussian blue method was preferred to the Turnbull blue. Lison's procedure of the former, slightly modified, gave the most satisfactory results. When a procedure is required that employs non-iron-containing reagents, Macallum's or Mallory's hema-toxylin and Quincke's ammonium sulfide method are useful. The former, though not entirely specific, is preferable under controlled conditions when the quantities of iron are small. Hemoglobin iron in paraffin sections can be demonstrated by the usual procedures for iron after previous exposure of the section to peroxide, as recommended by Brown. The property of nuclei to adsorb iron from inorganic sources and from hemoglobin can readily be shown; caution is required in interpreting the iron detected in nuclei after Macallum's sulfuric acid hydrolysis.     

Histochemical Tests for Polyphenols in Plant Tissues

A satisfactory histochemical test for polyphenols in fresh plant tissues is described. The test is based upon a colorimetric method for phenolics using a nitrous acid reaction. Certain catechol derivatives are characterized by the formation of an intense cherry-red in fresh sections of plant tissues with the reagents used. The red color is sufficiently intense to be readily observed microscopically at 100X. Results obtained with other tests also are described.     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

Acid Phosphatase in Dictyostelium discoideum

An acid phosphatase has been studied at various stages of development of Dictyostelium discoideum. This enzyme has a pH optimum of 3.6 and catalyses the hydrolysis of both hexose phosphates and mononucleotides as well as that of p-nitrophenyl phosphate. The enzyme specific activity increases 2–4 fold in vitro during differentiation, but the activity in vivo may be controlled through endproduct inhibition by orthophosphate, which accumulates in the cells during sorocarp formation. The predicted activity in vivo is highest in the middle of development, especially at the pre-culmination stage.     

Histochemical Demonstration of Protein-Bound Alpha-Acylamido Carboxyl Groups

A method has been developed to demonstrate the alpha-acylamido carboxyl groups of protein, taking advantage of the fact that acylamido carboxyl groups are converted to ketonic carbonyls by the action of acetic anhydride and absolute pyridine. The method utilizes deparaffinized sections of tissues fixed in a variety of fixatives. Following the conversion of carboxyls to the methyl ketones, the latter are stained with 2-hydroxy-3-naphthoic acid hydrazide. Control experiments have indicated that methylation of carboxyls prevented staining, as did carbonyl reagents after the carboxyls were transformed to methyl ketones. Leucofuchsin did not stain the ketonic carbonyls, and only elastic tissue stained with 2-hydroxy-3-naphthoic acid hydrazide without the previous use of the catalyzed reaction with anhydride. A brief survey of the reaction on various tissues of the albino rat was made, and the effects of various fixatives were assayed. Of particular interest were certain sites, such as acidophiles of the anterior pituitary gland, where an intense reaction occurred. The possibility exists that certain specific proteins rich in terminal acylamido carboxyl groups, by virtue of their protein side chains or low molecular weight, may be demonstrated by this method.     

Demonstration of Acid Phosphatase in Eimeria spp.: Partial Characterization of the Enzyme in E. vermiformis1,2

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporubted oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chroma-tography.     

Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

铅对鲫鱼碱性磷酸酶和酸性磷酸酶活性的影响

在实验条件下,将鲫鱼置于1、10、40 mg/L的Pb2 溶液中静态染毒,分别在24 h、48 h、96 h、144 h测定鳃、肝、胰、肾、脑组织的碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性.结果 表明,鲫鱼鳃、肝、胰、肾、脑组织中碱性磷酸酶和酸性磷酸酶的活性随着铅浓度的升高和染毒时间的延长均呈下降趋势,其中以肾脏和脑下降最明显.     

Histochemical Demonstration of Succinic Dehydrogenase in Ascites Tumor Cells

Fresh undiluted tumor ascites (0.05 ml) withdrawn from peritoneal cavity was placed immediately in a centrifuge tube containing 2.0 ml of an aqueous mixture prepared with 1 part each of the following solutions: 1% neotetrazolium chloride, 0.2 M sodium succinate and 0.1 M phosphate buffer, pH 7.4. The tube was incubated for 2 hr at 37°C and centrifuged for 3 min at 700 rev/min. The precipitate was washed with 0.85% saline solution and subsequently fixed with neutral 10% formalin for 10 min. After centrifugation, smears or squash preparations of the precipitate were prepared. Succinic dehydrogenase activity was demonstrated very distinctly and uniformly by the granular deposition of a deep purple pigment intracellularly.