Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

A new direct lead technique for histochemical demonstration of alkaline phosphatase activity.

A lead method for demonstrating alkaline phosphatase is described. The method is based on direct precipitation of lead as lead phosphatase at pH 9.5, the pH optimum of the enzyme. Stable incubation medium was achieved by using tartrate, instead of maleate, as chelating for lead. The method was found to be suitable for visualization of alkaline phosphatase in different types of tissues.     

Mouse ovarian alkaline phosphatase activities that respond to gonadotropins: histochemical and biochemical studies.

Alkaline phosphatase activity was measured in whole ovarian homogenates from pre-pubertal mice of different ages, with and without prior injection of human chorionic gonadotropin. Alkaline phosphatase activity was also scored in the different cell types in sections of similar ovaries, using two distinct histochemical procedures. The results from those methods differed. Biochemical studies indicated the presence of three distinct alakaline phosphatase activities: I and Ib, both optimal at pH 10.4 and with similar substrate requirements and inhibitor sensitivities (phosphatase I being characteristic of unstimulated ovaries and Ib of ovaries stimulated with human luteinizing hormone or human chorionic gonadotropin), and phosphatase II, optimal at pH 9.4, with different substrate requirements and inhibitor sensitivities. The differences observed using the histochemical procedures can probably be accounted for by the effects of different incubation conditions on the activities of these three enzymes.     

A direct lead technique for histochemical demonstration of leucocyte alkaline phosphatase activity in blood smears.

A histochemical technique for demonstrating leucocyte alkaline phosphatase activity (LAP), based on direct precipitation of lead phosphate at pH 9.5, is described. The effect of fixative, temperature and stability of the medium on the activity of the enzyme and stability of the colour reaction was thoroughly studied. Peripheral blood smears obtained from both normal humans and pathological cases were studied and the results were compared with these obtained by the azo-dye method.     

Some histochemical and biochemical ovservations on the lymphatic tissues of highly inbred white leghorns

Lymphatic tissues of inbred lines of White Leghorn chickens and 20-day embryos (inbreeding coefficient exceeds 95%) were used in the experiments, e.g. line 6 was susceptible, line 7 was resistant, and line 15 I was intermediate in response to the virus. Enzyme reactions were studied in cryostat-cut sections of tissues and in tissue minces by colorimetric procedures. Numbers of isozymes and proteins of lymphatic tissues were resolved by disc gel electrophoresis. Colorimetric tests showed that intensities of lactate, malate, isocitrate and succinic dehydrogenase catalyzed reactions were higher in the bursae of 15 I 20-day embryos than they were in bursae of either line 6 or 7 embryos. Intensity of dehydrogenase reactions of the spleen (15 I embryos) exceeded that found in line 6 and 7 embryos. Intensity of diaphorase reactions in the spleen and thymus was fairly uniform in all lines of embryos. Intensity of DPN diaphorase reactions in the bursae of line 15 I embryos exceeds that found in either line 6 or 7 embryos. Intensity of enzyme reactions leveled off to become fairly uniform in lymphatic tissues of chickens 3–4 weeks post hatching with the exception that dehydrogenase reactions were less intense in the thymus of 15 I chickens. Photodensitometer scans of acrylamide gel columns showed that proteins of line 6 lymphatic tissues combined with less Amido black 10B than lymphatic proteins of either line 15 I or 7 embryos. There was fairly good agreement between concentrations of strong mobility (components 1–9) and weak mobility (components 10–16) in lymphatic tissues of all lines of embryos with the exception that strong mobility proteins were about twice as concentrated in line 15 I bursae. Variable numbers of lactate isozymes were found in the lymphatic tissues of 20-day embryos.     

Quantitative histochemical and stereological study of alkaline phosphatase in the rat thyroid gland

Summary The histochemical alkaline phosphatase reaction in thyroid vascular endothelium was estimated spectrophotometrically and stereologically. Thus two main characteristics of the reaction were obtained: 1. the average enzyme activity, reflecting the level of transport processes in the capillary-thyrocyte system; 2. the relative volume of functioning vessels. An index of thyroid vascularity is proposed that is equal to the product of these two characteristics. The changes of the primary characteristics as well as of the vascularity index, caused by experimental hypo- and hyperplasia of the gland, are discussed.     

Distribution of xanthine oxidoreductase activity in human tissues--a histochemical and biochemical study.

Localization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly-(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.     

Comparative biochemical study of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals

Alkaline phosphatases from the liver, kidney and intestine in various vertebrates were strongly inhibited by beryllium, 2-mercaptoethanol, potassium cyanide and EDTA. The enzymes showed various sensitivities to the inhibition by zinc and to heat denaturation at 56 degrees C for 5 min at pH 7.0. The liver and kidney enzymes showed higher sensitivity to the inhibition by L-homoarginine than by L-phenylalanine. The intestinal enzymes in higher vertebrates were more sensitive to the inhibition by L-phenylalanine than by L-homoarginine, whereas the intestinal ones in lower vertebrates showed quite similar sensitivities to both amino acids.     

A rare electrophoretic enzyme variant of serum alkaline phosphatase in a group of Icelanders.

An electrophoretic isoenzyme variant of serum alkaline phosphatase was found in 10 out of 343 subjects belonging to an Icelandic population in Husavik and the Husavik region. 9 of the variant-positive subjects were women. The enzyme variant differs from normal isoenzymes in electrophoretic mobility, substrate specificity, and response to inhibitors. It could be demonstrated that nine of the subjects with the enzyme variant were related with each other.     

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

Summary The indoxyl—tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100mm Tris-HCl buffer, pH 9.0, 0.2–1.0mm substrate, 0.32mm 1-methoxyphenazine methosulphate, 10mm MgCl₂, 5mm sodium azide and 1mm tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1–2mm and 5mm respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 µm and therefore, the reaction obeyed the Beer—Lambert law. Variation of the substrate concentration yielded aK_M of 0.05mm for aqueous media and aK_M of 0.55mm for polyvinyl alcohol-containing media. The inhibition with tetramisole appeared to be competitive withK_i = 0.07mm for aqueous media andK_i = 0.7mm for polyvinyl alcohol-containing media. These values indicate that the indoxyl—tetranitro BT method is considerably more sensitive than any metal salt or diazonium salt method developed so far. It is concluded that the optimized method described here is a specific, sensitive and valid quantitative histochemical method for the demonstration of alkaline phosphatase activity.