Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN₃), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN₃ as a blocker of the respiratory chain was indispensible, because without NaN₃ most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Microphotometric determination of enzymes in brain sections. II. GABA transaminase

Summary The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM α-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 μm and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Microphotometric determination of enzymes in brain sections

Summary A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN₃, 10 mM MgCl₂, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 m. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1, 2-2)     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of lactate dehydrogenase activities in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of lactate dehydrogenase into the medium during incubation. In the histochemical system the NAD⁺-dependent enzyme catalyzes the electron transfer from lactate into NAD⁺. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Amytal is incorporated into the incubating-medium to block electron transfer to the cytochromes. Problems involved in the histochemical demonstration of lactate dehydrogenase activity are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Quantitative dehydrogenase histochemistry with exogenous electron carriers (PMS,MPMS, MB)

Summary The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0–8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt.Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higer SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Histochemical technique for the demonstration of pyruvate kinase activity

Summary We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP andd-glucose to yieldd-glucose-6-phosphate and ADP. Thed-glucose-6-phosphate is oxidized by exogenous and endogenousd-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP⁺, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Schurin azide and amytal are included to block electron transfei to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal arusele. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.Dedicafed to Professor Dr. T.H. Schicbler on the occasion of his 65th birthday     

Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles

Summary A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate d-fructose-6-phosphate to d-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into d-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the d-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-d-glycerate. Concomitantly the electrons are transported via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Meldola Blue: A new electron carrier for the histochemical demonstration of dehydrogenases (SDH,LDH, G-6-PDH)

Summary A new electron carrier, Meldola Blue (8-dimethylamino-2,3-benzophenoxazine; Boehringer Mannheim GmbH, Deutsche Patentschrift P 1959410) was tested for its usefulness in the histochemical demonstration of dehydrogenase activity in adrenal cortex, liver, heart muscle of guinea pig and human oviduct and compared with PMS.For demonstrating SDH activity Meldola Blue (MB) is as efficient as PMS. A decisive advantage of MB as compared with PMS is its low sensitivity to light exposure, facilitating direct visualisation of histochemical reaction processes.Generally, a high diffusion rate of reduced electron carriers (PMS and MB) from the section into the incubation medium (PVA) leads to a loss of reduction equivalents, particularly in the demonstration of NAD- or NADP-dependent dehydrogenases (LDH, G-6-PDH) with lower TNBT concentrations. However, no inhibition of SDH-, LDH- and G-6-PDH activities was observed with incubation media containing the tested concentrations of PMS and MB.     

On the subsarcolemmal localization of phenazine ethosulfate-linked alpha-glycerophasphate dehydrogenase activity in pigeon pectoralis white muscle fibres.

In this study frozen sections of avian striated muscles were incubated for mitochondrial alpha-glycerophosphate de hydrognease (alpha=GPD) reaction, and the effect of menadione, phenazine methosulfate (PMS) or phenazine ethosulfate (PES) as intermediate electron acceptors was evaluated. Under histochemical conditions, PMS or PES-linked alpha-GPD reaction was poor in the chicken posterior latissimus dorsi and chicken pectoralis muscles. However, PMS or PES-linked alpha-GPD reaction was present characteristically in the subsarcolemmal mitochondria of the "broad white" fibres of the pigeon pectoralis muscle only; the subsarcolemmal mitochondria of the narrow red fibres lacked such a reaction pattern. The above reaction pattern, however, differed when compared with the menadione-linked alpha-GPD reaction. The present histochemical evidence suggests the existence of an inherent heterogeneity in the mitochondrial populations of the different avian striated muscle fibres studied.     

Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary Improved histochemical multi-step techniques for the demonstration of glucose 6-phosphate isomerase and phosphoglucomutase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the glucosephosphate isomerase converts the substrate d-fructo-furanose 6-phosphoric acid to d-gluco-pyranose 6-phosphoric acid, and the phosphoglucomutase converts the substrate -d-glucose 1-phosphate to the same reagent, which in turn is oxidized, by exogenous and endogenous glucose 6-phosphate dehydrogenase to d-glucono--lactone 6-phosphoric acid. Concomittantly the electrons are transferred via NADP⁺, phenazine methosulphate and menadione to nitro-BT. Sodiumazide and amytal are incorporated to block electron transfer to the cytochromes.     

A histochemical and enzymatic study of the muscle fiber types in the water monitor, Varanus salvator

Histochemical analysis of five muscles from the water monitor, Varanus salvator, identified three major classes of fibers based on histochemical activities of the enzymes myosin ATPase (mATPase), succinic dehydrogenase (SDH), and alpha-glycerophosphate dehydrogenase (alpha GPDH). Fast-twitch, glycolytic (FG) fibers were the most abundant fiber type and exhibited the following reaction product intensities: mATPase, dark; SDH, light; alpha GPDH, moderate to dark. Fast-twitch, oxidative, glycolytic (FOG) fibers were characteristically mATPase, dark; SDH, light; alpha GPDH, moderate to dark. The third class of fibers had the following histochemical characteristics: mATPase, light; SDH, moderate to dark; alpha GPDH, light. These fibers were considered to be either slow twitch, or tonic, and oxidative (S/O). Pyruvate kinase (PK), alpha GPDH, and citrate synthase (CS) activities were measured in homogenates of the same muscles studied histochemically. There was a positive relationship between both PK and alpha GPDH activities and the percentage of glycolytic fiber types within a muscle. Likewise, CS activities were greater in muscles high in FOG and S/O content. Based on CS activities, Varanus S/O fibers were eight-fold more oxidative than FG fibers within the same muscle. PK/CS ratios suggested that FG fibers possess high anaerobic capacity, similar to the iguanid lizard Dipsosaurus. The fiber type composition of the gastrocnemius muscle, relative to that of other lizard species, suggests that varanid lizards may possess a greater proportion of FOG and S/O fibers than other lizards.     

A quantitative cytochemical study of UDP-d-glucose: NAD-oxidoreductase (E.C. 1.1.1.22) activity during stelar differentiation in Pisum sativum L. cv Meteor

Summary A quantitative cytochemical assay for UDP-d-glucose dehydrogenase (UDPGD) activity employing scanning and integrating microdensitometry has been revised and applied to a study of this enzmye during the initiation of secondary cell wall biosynthesis during the formation of primary vascular tissues in roots of Pisum sativum L. cv Meteor. The reaction involves the use of NBT as final electron acceptor and is inhibited 10-fold by either 10 mM UDP-d-xylose or 25 mM UDP-d-glucuronic acid, two molecules involved in feed-back inhibition of UDPGD activity in vivo. UDPGD is a far-from equilibrium enzyme representing a flux-generating step in the biosynthesis of precursors for hemicelluloses involved in secondary cell wall construction, and can be demonstrated to increase sharply in activity in cells of the developing primary vascular elements. This changed activity occurs 18–20 cells back from the root cap junction and coincides with the first cells containing the activated programme for secondary cell wall synthesis.     

Microphotometric determination of enzymes in brain sections. IV. Isocitrate dehydrogenases

A polyvinyl alcohol-(PVA) containing incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of NAD- and NADP-linked isocitrate dehydrogenases (ICDHs) in cryostat sections of the rat hippocampus. The following incubation medium is recommended for the quantification of NAD- and NADP- (differences in brackets) ICDHs: 100 mM DL-isocitrate, 10 mM sodium azide, 5 mM (4 mM) nitroblue tetrazolium (NBT), 7 mM NAD (4 mM NADP), 10 mM magnesium chloride, 0.25 mM phenazine methosulfate (PMS), with or without 5 mM ADP (without ADP), 23% PVA in 0.05 M Hepes buffer; the final pH was 7.5. With these incubation media a linear response of the reactions lasted at least 20 min. In kinetic and end-point measurements the same level of activities was demonstrable. The use of NaN3 (as a blocker of the respiratory chain) and PMS (as artificial electron carrier) was indispensible for the transfer of all reduction equivalents in the dehydrogenase reactions to the tetrazolium salt NBT. Furthermore, the activation by magnesium ions and the need of PVA to avoid diffusion artefacts of the loosely bound ICDHs were clearly shown. It is concluded that the quantification of ICDHs in situ could be a valuable tool for neurochemical investigations because ICDHs play a role not only in the substrate flux through the tricarboxylic acid cycle but also in providing alpha-ketoglutarate for the formation of glutamate which is an important amino acid in the brain.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Summary Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3–6 months old) and aged male rats (24–27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1 the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio entorhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Deutsche Forschungsmeinschaft (Ku 541/2-1)     

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.     

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme

Summary A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mm inosine, 0.47 mm methoxyphenazine methosulphate and 1 mm Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section was allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mm p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.     

Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry

The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences fo formalin fixation, acetone, magnesium ions, cyanides, electron carries (phenazine methosulfate, menadione coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity: Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon. Incubation of 5 micrometer cryostat sections in narrow jars in the following medium (38.5 ml):--10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).--18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).--2.6 mg KCN in 16 ml aqua bidest. (1 mM).--540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).--3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM). The incubation medium has an osmolarity of 440 mosm. The incubation is carried out for 10 min at 37 degree C in darkness. To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at--22 degree C or--40 degree C for 7--10 min and an incubation time of 20--30 min is recommended. Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.     

Improvement in Soluble Dehydrogenase Histochemistry by Nitroblue Tetrazolium Preuptake in Sections: A Qualitative and Quantitative Study

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/ acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.