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.     

Suitability of the azocoupling reaction with 1-naphthyl-beta-D-glucoside for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) in human enterobiopsies.

The suitability of the simultaneous azocoupling reaction with 1-naphthyl-beta-D-glucoside and hexazonium-p-rosanilin in the detection of the activity of lactase (or lactase-beta-glucosidase complex) in jejunal biopsies of patients with various forms of the malabsorption syndrome was tested. Results were compared with those obtained with the indigogenic method using 4-Cl-5-Br-3-indolyl-beta-D-fucoside which is the method of choice. Both methods gave identical results as far as the relative intensity of the brush border staining was concerned. The azocoupling method applied in unfixed cold microtome sections can be recommended for the routine diagnostics of the malabsorption syndrome when the indolyl substrate is not available.     

The histochemical demonstration of calcium with 8-hydroxyquinoline

Summary A method for the histochemical demonstration of calcium using 8-hydroxyquinoline was investigated. It was found to give intense, highly selective staining of various pathologic and natural calcifications, and compared favourably with conventional procedures for calcium. Calcium oxalate and calcium pyrophosphate were not stained.     

The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide

Summary The indigogenic method for aminopeptidase of Pearson et al. (1963) was critically evaluated. The localization obtained with it is not correct due to diffusion artifacts. Ferricyanide cannot be used as an oxidation agent. Based on experiments with other oxidation agents (phenazonium methosulfate, nitro BT, tetranitro BT) a new method was devised.The recommended incubation medium contains 0.9 mM L-N-(5-bromoindol-3-yl) leucinamide hydrobromide (chloride), 0.73 mM tetranitro BT, 0.27 mM phenazonium methosulfate and 0.67 M phosphate buffer pH 7.4. The enzyme activity is indicated by the deposition of tetranitro BT formazan.Results with this method in rat kidney, jejunum, liver, lung, and submaxillary gland, in monkey kidney and jejunum, and in human jejunal biosies are almost identical with those obtained with L-leucyl-4-methoxy--naphthylamide applied in a simultaneous azocoupling procedure. The given principle of the demonstration of aminopeptidase activity with an indolylamine substrate deserves a further exploration in the demonstration of peptidases in situ both on optical as well as electronmicroscopical levels.Dedicated to the 60th birthday of Prof. MUDr. J. vejda, DrSc., Head of the Department of Pathology, Medical Faculty, Purkyn University, Brno.     

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.     

Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.     

The histochemical demonstration of phosphoglucose isomerase

Summary A technique for the histochemical demonstration of phosphoglucose isomerase, using an indirect tetrazolium method, is described. The enzyme is shown to be widely distributed, thus confirming biochemical findings. The wide distribution is of significance because phosphoglucose isomerase occupies a position of considerable importance in carbohydrate metabolism, particularly in the conversion of fructose to glucose by means of intermediate phosphates.     

The histochemical demonstration of calcium with 8-hydroxyquinoline.

A method for the histochemical demonstration of calcium using 8-hydroxyquinoline was investigated. It was found to give intense, highly selective staining of various pathologic and natural calcifications, and compared favourably with conventional procedures for calcium. Calcium oxalate and calcium pyrophosphate were not stained.