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     

Fluorescence of indolylethylamines condensed with formaldehyde

Summary The reaction between some indolylethylamines and formaldehyde has been studied in model protein layers. 6-Hydroxytryptamine and 5,6-dihydroxytryptamine give a specific formaldehyde-induced fluorescence similar to that of 5-hydroxytryptamine and tryptamine with an emission peak in the region of 490–525 m. Although each substance has its specific fluorescence spectrum, it is not possible to safely differentiate them visually. They can, however, be distinguished by the use of microspectrophotofluorimetry. The formaldehyde-induced fluorescence of 6-hydroxytryptamine has a fluorescence intensity about four times greater than that of 5-hydroxytryptamine, and is in the same order of magnitude as that of noradrenaline.Abbreviations Used 5-HT 5-hydroxytryptamine - -m-5-HT -methyl-5-hydroxy-tryptamine - 6-HT 6-hydroxytryptamine - 5,6-diHT 5,6-dihydroxytryptamine - NA noradrenaline - T tryptamine     

The histochemical demonstration of peptidases by natural substrates

Summary Methods for the demonstration of peptidase activities in situ using natural peptides as substrates are described and evaluated. They are based on the use of the l-amino acid oxidase of Vipera ammodytes venom (or of similar snake venom) which is able to catalyze efficiently the oxidation of Leu, Met, Phe, Ileu, Tyr, and Try set free by the action of the particular peptidase(s). Accordingly, peptides from which the mentioned aminoacids are set free can be used as substrates. Cryostat sections adherent to slides are employed which can be preextracted with chloroform-acetone mixture (, 4°C, 5 min) to eliminate lipids. All substances needed for this multistep reaction are prepared and applied in a suitable gel layer which impedes the diffusion of soluble peptidases and also of l-amino acids set free by their action. The enzyme activity is assessed from the difference between the coloration of a pair of parallel sections one of which was incubated with the substrate and the other without it.There are two possible methods: a) The method using l-amino acid oxidase-phenazonium methosulphate and NitroBT (AAO-PMS-NBT method) works in the pH range 6.5–8 and requires anaerobic conditions. The medium consists of buffered substrate, Vipera ammodytes venom, PMS, NBT and KCN and is prepared preferentially in 10% gelatine. It is allowed to set on coverslips which are picked up by slides with sections and incubated at room temperature in the dark. In addition to a quick information on the firmness of the structure association of the peptidase splitting the substrate and on the influence of the fixation on its activity the method enables its cellular localization. Microdensitometric evaluation of formazan deposition during incubation is possible (there is a linear increase in density at least up to 20 min of incubation). b) The method using l-amino acid oxidaseperoxidase-3,3-diamino benzidine hydrochloride (AAO-PO-DAB method) works in the pH range 6–9 and requires aerobic conditions. The medium consists of buffered substrate, Vipera ammodytes venom, PO and DAB and is prepared preferentially in 1% agar solution which is applied on sections either directly or as a sandwich after gelification on a slide. The incubation proceeds at 37°C. The method furnishes similar information as the preceding one, however it is less sensitive and enables histological localization only.The suitability of these methods was shown among others on the demonstration of peptidases of the gastrointestinal tract and of the arterial wall. Biochemical findings reported so far were not only confirmed but entirely new data ascertained.In the human and rat intestinal mucosa the bulk of activities directed towards dipeptides (Leu-Leu, Leu-Gly, Gly-Leu, Ser-Met) is soluble. Peptidases splitting Leu-Leu-Leu are somewhat more structure bound in the human than in the rat intestine. Activities of these peptidases which are present even in crypt enterocytes increase during the process of the differentiation of enterocytes so that enterocytes covering sites and tops of villi display the highest staining intensity. The contribution of cells of the propria to the overall activity of the mucosa of the small intestine is not decisive. As against aminopeptidases A and M, dipetidyl peptidase IV and endopeptidase enzymes splitting natural di-and tripeptides can be demonstrated also in the gastric epithelium. However, in enterocytes the corresponding activity (activities) is (are) substantially higher already in the duodenal bulb. The differences in the staining intensity between gastric epithelium and enterocytes are lower in the case when Ser-Met is used as substrate than in the case when Leu-Leu; Gly-Leu, Leu-Gly and Leu-Leu-Leu are used as substrates. In patients suffering coeliac sprue the activity of peptidases residing in enterocytes splitting the substrates given above is lower in the acute stage of the disease than in normal persons. However, these soluble peptidases seem to be affected less than the brush border peptidases (in decreasing order): endopeptidase, -glutamyl-transferase, dipeptidyl peptidase IV, aminopeptidase A and aminopeptidase M.In the human aortae the highest activity was recorded using Ser-Met and Leu-Met as substrates, followed (in the decreasing order) by Leu-Leu, Leu-Leu-Leu, Met-Met-Met, Met-Val and Ser-Leu-Leu. No activity was recorded when N-CBZ-Leu-Met was used as the substrate. In normal human aorta the highest activity resides in vasa vasorum followed by muscle cells of the media. In cells of atherosclerotic plaques the activity is usually lower, except in cells of fatty streaks and small lipoid plaques where the activity can surpass that found in other cells of the aorta.     

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.     

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.     

The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide.

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-beta-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.     

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.     

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 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