Observations on the use of Nitro Blue tetrazolium in the detection of early myocardial changes

Synopsis Reserpine-induced cardiomyopathy in guineapigs was studied by means of histochemical dehydrogenase reactions using succinate as substrate and tetrazolium salts as electron acceptors. Succinate: Nitro Blue tetrazolium oxidoreductase activity in the ventricular muscle of reserpine-treated animals was localized cytochemically in a blue granular pattern instead of the purple myofibrillar pattern typical of the normal myocardium. A series of experiments designed to investigate their mechanism showed that the blue granular pattern is a physicochemical sequel secondary to the fatty deposition in the disordered myocardial cells and, moreover, the preferential deposition of the blue dinitroformazan is not indicative of augmented succinate: Nitro Blue tetrazolium oxidoreductase activity. It is concluded that the histochemical reaction for this enzyme is a very sensitive index of myocardial alterations associated with an early deposition of fat, not easily detectable by organotropic dye techniques.     

A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases

Summary A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but -OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results.     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Subgrouping of fast twitch fibres in skeletal muscles of man

Summary Subgroups of fast twitch (FT) muscle fibres were identified by histochemical techniques on muscle samples of m. quadriceps femoris from six male and six female subjects, who had been assigned to three groups; untrained, active and well trained (endurance runners). Slow twitch (ST) and FT fibres were initially identified using the histochemical stain for myofibrillar ATPase, preincubated at pH 10.3 and 4.3. Three people, working independently, then identified the subgroups FTa and FTb on the basis of the staining intensity for only one of the following reactions: -glycerophosphate dehydrogenase, -GPD; NADH tetrazolium reductase, NADH-TR; and myofibrillar ATPase preincubated at pH 4.6, ATPase (4.6). FTa fibres were clearly distinguished from the darker staining FTb fibres using the ATPase (4.6) reaction. Differences in the staining within the FT fibres using the -GPD and NADH-TR reactions were more subtle, and differences between subject groups were evident. The percentage of FTa fibres was overestimated for the untrained and underestimated for the well trained subjects using NADH-TR. With the -GPD stain the percentage of FTa fibres was generally underestimated. When the data for all three stains were compared, only 27% of the FT fibres were placed in the same subgroups. These results demonstrate that the subgrouping of FT fibres in man is more reliable using the differences in pH sensitivity for the myofibrillar ATPase reaction compared to histochemical reactions for oxidative or glycolytic enzymes.     

A tetrazolium method for non-specific alkaline phosphatase

Summary A technique for the histochemical demonstration of non-specific alkaline phosphatase was developed using a medium containing indoxyl phosphate and a tetrazolium salt, Nitro B.T. The tetrazolium salt was reduced to diformazan by the hydrogen ions released by the formation of either indigo or indigo white by reaction of the enzyme on the indoxyl phosphate.The localization in the organs investigated was similar to that obtained by the standard azo dye and lead techniques.     

Indoxyl alfa-D-galactoside as the temporarily last substrate for glycosidase histochemistry. The present state of the art in histochemical glycosidase research using indoxyl glycosidas

Initiated by the recently published histochemical method for the investigation of alfa-D-galactosidas with an indoxyl substrate, the current state of this group of synthetic compounds in light and electron microscopic histochemical glycosidase research is evaluated whereby historical, functional, methodological and applied aspects are considered. Beginning with the introduction of indoxyl acetate for non-specific esterase in 1951 and 1952 numerous other indoxyl substrates and mostly substituted in the 5- and 4-position of the indol ring by Br and Cl were developed to study histochemically non-specific phosphatases and glycosidases and frequently used in indigogenic, azoidoxyl, tetrazolium salts and metal salt techniques for catalytic (activity) histochemical and less often for immunohistochemical, affinity histochemical and hybridohistochemical purposes. The last substrate which became available and was validated for activity histochemistry was 5-Br-4-Cl-3-indoxyl alfa-1-galactoside for alfa-1-galactosidase. At present, the indoxyl glycosides are more widely used than 5-Br-4-Cl-3-indoxyl acetates and phosphates when compared with the alternative synthetic (artificial) naphthol, 6-Br-2-naphthol or ternative synthetic (artificial) naphthol, 6-Br-2-naphthol AS substrates, and among the indoxyl glycosides those for the oxoglycosidases lactase, maltase-glucoamylase, glucoamylase, acid beta-D-galactosidase, neuroaminidase and alfa-D-galactosidase are superior to other artificial compounds. When one considers in addition, electron microscopic catalytic glicosidase histochemistry (ultracytochemistry, 5-Br-4-Cl-3-indoxyl is the only suitable moiety for this purpose. These glycosidase can mostly be localized in plasma membranes or lysosomes and also measured there in tissue sections but are also found in secretion granules, endoplasmic reticulum and organ lumina.     

Selection of optimum tetrazolium salts for use in histochemistry: The value of structure-staining correlations

Summary A structure-staining correlation study was made of a number of tetrazolium salts and formazans used in histochemistry. Numerical parameters describing molecular structure were calculated from structural formulae. Staining data were from previously published work. It was found that several significant practical variables (namely, uptake and reducibility of tetrazolium salts; substantivity, lipid solubility, and crystal size of formazans) could be predicted from structural parameters. Possible applications of these correlations are discussed: to increase our understanding of existing staining systems and to guide the design and selection of new tetrazolium salts for histochemical use.     

Succinic dehydrogenase activity in liver,kidney and brain of rat

Summary A method is described for histochemical quantification of the activity of succinic dehydrogenase in various tissues of rat by means of Nitroblue tetrazolium. This method can be used for comparison of enzyme activities; the activities calculated correspond to values obtained by biochemical methods. The necessity to quantify the nothing dehydrogenase is established as well as the amount of half-formazan.Accepted as doctoral dissertation by the Faculty of Medicine, Westfälische Wilhelms-University Münster     

Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 m in thickness. For all the enzymes that could be detected, the 6 m : 3 m ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.     

Quantitative aspects of the histochemical tetrazolium salt reaction on monoamine oxidase activity in rat liver

Summary The tetrazolium method for the histochemical detection of monoamine oxidase (MAO) activity in rat liver cryostat sections has been tested for its specificity and its possible use in quantification. The tetrazolium salt tetranitro blue tetrazolium is recommended for the localization of MAO activity, rather than nitro blue tetrazolium or BPST [2-(2-benzothiazolyl)-3(4-phthalhydrazidyl)-5-styryl-tetrazolium]. Hardly any formazan was produced in the absence of the substrate tryptamine and Marsilid, a specific inhibitor of MAO activity, prevented formazan production almost completely. A linear relationship between the integrated absorbance measured with a microdensitometer and either the incubation period or section thickness was obtained. We conclude that the method described in this paper can be used for the quantitative analysis of MAO activity in tissue sections of rat liver. MAO activity was found to be 20–25% higher in the periportal zone of rat liver than in the perivenous zone.     

Improved cytochemical methods for demonstrating cell death using LR White as an embedding medium

A series of techniques based on LR White resin are described, which permit the use of an anti-histone antibody for the in situ localization of DNA fragmentation characteristic of apoptosis at both the light and the electron microscope level. The methods, applied to an untreated squamous carcinoma of the pharynx, allow direct comparison of light microscopic localization of exposed nucleosomal histones using 3,3-diaminobenzidine (DAB) and silver-enhanced techniques with a colloidal gold-based anti- histone technique at the electron microscope level. Parallel histochemical localization of acid phosphatase activity is also presented. © 1998 Chapman & Hall     

The demonstration of mouse lung lactate-yellow tetrazolium reductase

Summary Yellow tetrazolium has been examined for use as a qualitative histochemical reagent in light microscopy. Experimental conditions for the demonstration of the lactate-yellow tetrazolium reductase system in mouse lung are described, and the possibility of subsequently demonstrating either NADH-MTT reductase or 3-glycerophosphate-MTT reductase in the same section has also been investigated. The yellow colour of the deposited formazan is uniquely favourable for counterstaining with blue nuclear stains.This paper forms part of a thesis for the Ph. D. degree submitted to the Council for National Academic Awards by J.E.E.     

Oxidative enzymes (tetrazolium reductases) in the hippocampal region of the rat

Summary This study deals with the activity and distribution of the following tetrazolium (Nitro-BT) reductases in the hippocampal region of the rat: NADH-,NADPH-, -glycerophosphate-, succinate-, the NAD⁺-linked lactate-, malate-, isocitrate-, glutamate-, -HO-butyrate-, the NADP⁺-linked glucose-6-phosphate-, malate-, and isocitrate tetrazolium reductases. Several architectonic pictures of enzyme distribution were obtained, the number of which was further increased by adding to the incubation media phenazine methosulfate, which is supposed to function as an electron transport agent.The laminar patterns of the various enzymes are described and commented on for each cortical area (p. 173). Then the patterns of parcellation resulting from the changes of laminar patterns are presented (p. 198). There is an excellent agreement between enzyme distribution and previously established architectural landmarks.Finally some aspects of the observations are discussed and correlated to earlier enzyme histochemical and neurobiochemical studies.Supported by the Norwegian Research Council for Science and the Humanities and by The National Institute of Neurological Diseases and Blindness, U. S. Public Health Service, Grant NB 02215. This aid is gratefully acknowledged.     

Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases

Summary In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, -glucuronidase, non specific arylesterase, microsomal arylsulphatase, -galactosidase, -N-acetylglucosaminidase, acid -glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

Summary New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca-and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H₂O₂ which decomposes at 55°–60° C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H₂O₂ mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H₂O₂-DAB and especially the H₂O₂-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.     

Histochemistry for nanomedicine: Novelty in tradition

During the last two centuries, histochemistry has provided significant advancements in many fields of life sciences. After a period of neglect due to the great development of biomolecular techniques, the histochemical approach has been reappraised and is now widely applied in the field of nanomedicine. In fact, the novel nanoconstructs intended for biomedical purposes must be visualized to test their interaction with tissue and cell components. To this aim, several long-established staining methods have been re-discovered and re-interpreted in an unconventional way for unequivocal identification of nanoparticulates at both light and transmission electron microscopy.Key words: histochemistry, immunohistochemistry, nanoparticles, light microscopy, transmission electron microscopy     

Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines

Summary Glyoxylic acid vapour is a most powerful reagent for the fluorescence histochemical visualization of biogenic monoamines. In the present investigation the mechanisms of fluorophore formation in the glyoxylic acid reaction has been studied in detail for tryptamine in histochemical models and in freeze-dried tissue, utilizing microspectrofluorometric, Chromatographic, and mass spectrometric techniques in combination with isotope measurements.The glyoxylic acid-tryptamine reaction proceeds through an initial Pictet-Spengler type cyclization to 1,2,3,4-tetrahydro--carboline-1-carboxylic acid, followed by two alternative fluorophore forming reactions yielding 3,4-dihydro--carboline, or the 2-carboxymethyl-3,4-dihydro--carbolinium and 2-methyl-3,4-dihydro--carbolinium salts, which are all strongly fluorescent. It is shown that the yield of fluorophores is considerably higher in the glyoxylic acid vapour reaction than in the formaldehyde vapour reaction of the standard Falck-Hillarp method, and that this higher efficiency of glyoxylic acid is due to the most favourable catalysing properties of the carboxylic group of the glyoxylic acid molecule.     

A comprehensive characterisation of the fibre composition and properties of a limb (<Emphasis Type="Italic">Flexor digitorum superficialis,membri thoraci</Emphasis>) and a trunk (<Emphasis Type="Italic">Psoas major</Emphasis>) muscle in cattle

Background  

The fibre type attributes and the relationships among their properties play an important role in the differences in muscle capabilities and features. Comprehensive characterisation of the skeletal muscles should study the degree of association between them and their involvement in muscle functionality. The purposes of the present study were to characterise the fibre type composition of a trunk (Psoas major, PM) and a limb (Flexor digitorum, membri thoraci, FD) muscle in the bovine species and to study the degree of coordination among contractile, metabolic and histological properties of fibre types. Immunohistochemical, histochemical and histological techniques were used.