Polyacrylamide gel technique for the histochemical demonstration of soluble enzymes.

Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliary enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied. To demonstrate the suitability of the method glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucomutase and aldolase were visulaized in human or rat skeletal muscle. Cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase were both visualized in the absence of added NAD+ and menadione. For the visualization of ATP producint enzymes, like creatine kinase and pyruvate kinase, the method is not suitable.     

Decalcification for histochemical demonstration of hydrolytic and oxidative enzymes

Summary A method for histochemical demonstration of hydrolytic and oxidative enzymes following decalcification was described in mature bone and tooth by neutral EDTA decalcifying solution.The decalcifying solution, 0.5 M EDTA tetrasodium salt was adjusted to neutral pH with 5 M citric acid, obtained the most sufficient results for demonstration of enzymes in decalcified tissue. The hard tissue decalcified in 30 to 40 days at 4° C exhibited a good preservation of hydrolytic and oxidative enzymes histochemically, but a few structural destruction in a long term decalcification was found in soft tissues and certain organs.     

The histochemical demonstration of choline acetyltransferase by semipermeable membrane technique

Summary The application of the semipermeable membrane technique in light microscopical demonstration of choline acetyltransferase is described. The method founds upon earlier developed lead salt techniques. Use of semipermeable membranes fully prevents any loss of enzyme by dissolvement or inactivation during fixation. Addition of NaCl to the incubation medium markedly increases the activity of choline acetyltransferase.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

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.     

Lectins for histochemical demonstration of glycans

Lectins have been proven to be invaluable reagents for the histochemical detection of glycans in cells and tissues by light and electron microscopy. This technical review deals with the conditions of tissue fixation and embedding for lectin labeling, as well as various markers and related labeling techniques. Furthermore, protocols for lectin labeling of sections from paraffin and resin-embedded tissues are detailed together with various controls to demonstrate the specificity of the labeling by lectins.     

The histochemical demonstration of five lysosomal enzymes in the pars distalis of the amphibian pituitary

Summary The distribution of five lysosomal enzymes (acid phosphatase, -glucosaminidase, -glucuronidase, sulphatase and E 600-resistant esterases) was studied in the pars distalis of the urodelian pituitary. Naphthol-AS-compounds coupled with hexazonium-pararosanilin yielded particularly good localization. The typical lysosomal picture and a basically similar distribution were obtained throughout. The reaction product was mainly found in the globules of the globular basophils (-cells) reported as gonatropin producing cells by several authors. Some speculations on the possible function of the high amount of the lysosomal enzymes in the globular basophils are made.Dedicated to Professor W. Bargmann on his 60th birthday.This work was partly supported by a grant from Deutscher Akademischer Austauschdienst, Bad Godesberg.     

A contribution to the histochemical demonstration of some hydrolytic and oxidative enzymes in plants

Summary In this work an attempt was made to apply to plant material some recent methods for studying enzymes in situ enabling even the intracellular localization. The following enzymes were demonstrated in the root tip of the broad bean: acid phosphatase, non-specific esterase, DPN tetrazolium reductase and the system of lactic acid dehydrogenase. The attempts to reveal the alcaline phosphatase were not successful even in unfixed material. A comparison was made of different techniques, their advantages and/or possible sources of errors were discussed. With fixed material satisfactory results were obtained.With 7 Figures in the Text, of which 4 in Colour     

Fluorescence methods for the histochemical demonstration of monoamines

Summary The histochemical fluorescence method of Falck and Hillarp for the demonstration of catecholamines and certain tryptamines, e.g. 5-hydroxytryptamine is based on the principle that these amines can be condensed with formaldehyde to yield strongly fluorescent 6,7-dihydroxy-3,4-dihydroisoquinolines and 6-hydroxy-3,4-dihydro--carbolines respectively. The investigation here reported presents the fluorescence characteristics and relative fluorescence yields for formaldehyde treated biogenic monoamines and certain related compounds enclosed in a dried protein layer. The fluorescence properties of some synthetic 6,7-substituted-3,4-dihydroisoquinolines and 3,4-dihydro--carbolines are given, and the fluorescence characteristics in relation to the molecular structure are discussed.Abbreviations used A adrenaline - DA dopamine - DOPA 3,4-dihydroxyphenylalanine - DOPS 3,4-dihydroxyphenyl-serine - 5-HT 5-hydroxytryptamine - 5-HTP 5-hydroxytryptophan - 5-MT 5-methoxytryptamine - -m-DA -methyl-dopamine - -m-DOPA -methyl-3,4-dihydroxyphenylalanine - -m-NA -methyl-noradrenahne - MTA 3-methoxy-tyramine - NA noradrenaline - NM normetanephrine - T Tryptamine - Try Tryptophan     

A device for the retrenchment of coupling enzymes for demonstration of creatine kinase on polyacrylamide gel.

We have established several optimal conditions for qualitative and quantitative allantoin determination by applying Ehrlich's reagent. The limit of detection for allantoin determination amounts to 5 × 10^?6 mm. Allantoin is determined quantitatively by measuring the absorbance at 440 nm (from 300 to 1000 μg/ml). The color of the complex becomes stable by standing for 10 min at room temperature. We have used these conditions for allantoin determination in Agrostemma githago seed.     

A substrate-film method for the histochemical demonstration of cellulase

Summary A substrate-film method has been devized for the histochemical demonstration of cellulase. The substrate film is made of sodium carboxymethyl-cellulose, which is made insoluble in water by fixation in acid ethanol. The tissue is briefly fixed in cold formalin, washed, and sectioned with a freezing microtome. The sections are mounted on slides, and covered with a piece of carboxymethyl-cellulose film, and the slide is incubated in a warm, moist atmosphere. After incubation, the film is stained with toluidine blue, and sites of cellulase activity appear as pale or clear patches in the film.In the digestive systems of certain molluscs, cellulase has been found in the lumens of the crop and stomach, and in the lumen and absorptive cells of the digestive gland tubules. The salivary glands, and the epithelia of the crop and stomach, show no reaction.Sections of control tissue, inactivated by boiling in water, do not show any reaction.I thank the Nobel Division of Imperial Chemical Industries Ltd. for information on their product Cellofas B 10, and for permission to publish that information.     

A histochemical method for the demonstration of unsaturated lipids

Summary A method has been developed for the histochemical demonstration of unsaturated lipids in light microscopy. It is a peracetic acid-thiocarbohydrazide-silver protein sequence followed by a physical development procedure. In the present study on paraffin and cryostat sections of liver, brain and ovary, unsaturated lipids were visualized as distinct reaction products coloured various shades of brown and black. The reaction products are easier to see and the method is more efficient than the peracetic acid-Schiff method.