Nitroblue Tetrazolium Method Research Articles

Multiple Use of Blotted Polyvinylidene Difluoride Membranes Immunostained with Nitro Blue Tetrazolium

A simple procedure for reusing polyvinylidene difluoride membranes with bound proteins reacted with antibodies and developed by the nitro blue tetrazolium method (M. S. Blake, K. H. Johnston, G. J. Russell-Jones, and E. C. Gotschlich (1984) Anal. Biochem. 136, 175-179) is described. The blue diformazan precipitate produced by the reduction of the nitro blue tetrazolium salt was dissolved by soaking the membrane in dichloro- or trichloromethane for 10-60 min at room temperature. After equilibration in methanol and then in distilled water the first and second antibodies were removed by incubating the membrane in 0.2 M glycine-HCl buffer, pH 2.2, for 90 min at room temperature. These cleaned membranes could be successively reused with different monoclonal antibodies or preimmune serum up to 21 times. The intensity of the spots or electroblotted bands and their specific reaction pattern with different monoclonal antibodies remained unchanged, and no background was observed when the membranes were reacted with preimmune serum. This procedure makes it possible to reuse membranes freshly developed or stored for long periods without loss of resolution.

Scavenged Effects of Cu(II) Chelates of Imidazole and Thiazole Derivatives on the Active Oxygen Species in vitro.

Scavenged effects of Cu (II) chelates of imidazole and thiazole derivatives on superoxide (SO), H2O2 and OH radical were studied and compared with that of cimetidine, which is well known as a superoxide scavenger, and with those of superoxide dismutase (SOD), catalase and Cu (ClO4)2. Six of imidazole and 6 of thiazole derivatives were cimetidine and famotidine analogues, respectively. Superoxide scavenged effects (IC50) of Cu (II) chelates were in the range of 0.48-5.67μM determined by the nitroblue tetrazolium method in a phenazine methosulfate-NADH system (method A) and in a xanthineoxidase-hypoxanthine system (method B), whereas the IC50 values of SOD were 4.5×10-2 and 3.4×10-2μM by the methods of A and B, respectively. No catalase activity of Cu (II) chelates were observed. In the studies of the scavenged effect of Cu (II) chelates on hydroxyl radical, IC50 values of Cu (II) chelates of N-methyl-N'-[2-(imidazol-4-yl) methylthioethyl]-thiourea (I-1) and its 5-methylimidazole derivative (I-2), which possessed methylthiourea groups, were 13.7 and 8.0μM, respectively, and the Cu chelate of I-2 had the highest scavenged activity against hydroxyl radical generated from Fenton reaction.

Serum superoxide dismutase in patients with Graves' disease

We studied serum superoxide dismutase (SOD) in patients with Graves' disease. Measurements of immunoreactive Cu, Zn-SOD and SOD-like activities were made by enzyme-linked immunosorbent assay (Ube Industries Ltd) and Nitroblue Tetrazolium method (Wako Ltd), respectively. Serum from patients with untreated Graves' disease had a significantly higher concentration of Cu, Zn-SOD and higher SOD-like activity than those from normal subjects, patients with Graves' disease under treatment over one year, patients with Graves' disease in remission, and patients with untreated Hashimoto's disease. Methimazole treatment produced no significant change in SOD-like activity and Cu, Zn-SOD concentration when patients with Graves' disease had normal thyroid function. These results indicated that oxidative tissue injury existed in patients with Graves' disease with normal thyroid function under treatment; however, we could not clearly establish that thyroid function had a direct connection with oxidative tissue injury.

A New Technique for Affixing Fresh Frozen Sections to Cold, Untreated Glass Slides and its Application to Histochemical Localization of Low-Km Aldehyde Dehydrogenase Activity, Inhibited by Cyanamide, in Mouse Liver.

A simple procedure, called the “cold acetone (-70°C) -dropping affixation technique”, for affixing fresh, frozen sections to cold, clean, untreated glass slides is described. A few drops of absolute acetone (-70°C) were dropped with a precooled pipet to the sections placed on the slides, and the acetone on the slides was absorbed immediately with a cold filter paper. The slides with the sections were left in a cryostat for approximately 30 min, so that the sections were affixed firmly to the slides. By this method we could prevent the diffusion of low-Km aldehyde dehydrogenase (ALDH) from the sections.The localization of low-Km ALDH activity in the hepatic lobule of normal mice was studied histochemically with the nitroblue tetrazolium (NBT) method. Reaction products, purplish-blue formazan showing the enzymatic activity, were found mainly in the centrilobular zone. Cyanamide (5 mM) inhibited the low-Km ALDH activity, whereas the high-Km ALDH activity was unaffected even by 10 mM cyanamide.

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Repeated,in vivo observation of frog neuromuscular junctions: remodelling involves concurrent growth and retraction

The fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide was used as a vital stain to study remodelling of motor nerve terminals in sartorius muscles of living frogs (Rana pipiens). Identified terminals were observed twice in vivo at intervals of 87-192 days. After the second observation, muscles were fixed and stained with the nitroblue tetrazolium method for nerve terminals and with cholinesterase stain. Observations were made of 243 junctions in 26 frogs. Most nerve terminals grew during the observation interval, with an average increase in total terminal length of 29%. This growth involved substantial remodelling. Within single junctions, the change in size was the net result of differing degrees of growth or shrinkage in individual nerve terminal branches. At least one new terminal branch appeared in 25% of the junctions. Terminal retraction was also common, with branch shortening seen in 60% of junctions and the complete disappearance of a branch in 12%. In one case the original axonal input retracted completely and the junction was partially reinnervated by a terminal sprout from a junction on an adjacent fibre. Some discrepancies between histological and in vivo observations of remodelling were noted. These observations confirm that frog neuromuscular junctions are highly dynamic synapses, subject to profound structural remodelling throughout adult life.

Double staining procedure for histochemical localization of alcohol and aldehyde dehydrogenase activities in the mouse liver.

The double staining procedure using both the nitroblue tetrazolium (NBT) and copper ferrocyanide methods was presented for the simultaneous localization of two different dehydrogenase activities in a single histologic section.Sections of normal mouse liver were stained for alcohol dehydrogenase (ADH) activity using the NBT method first, then stained for aldehyde dehydrogenase (ALDH) activity using the copper ferrocyanide method. However, the formazan showing ADH activity must chelate metal ion (Cu2+, Co2+, or Ni2+) prior to staining for ALDH activity.Both the blue metal-formazan complex showing ADH and brown copper ferrocyanide showing ALDH activities in a single liver section were localized mainly in the perivenous zone of the hepatic lobule. In addition, ALDH activity is always localized in the zone of the lobule where ADH activity is present.

Assay of superoxide dismutase activity in animal tissues

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine-xanthine oxidase ferricytochromec, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochromec method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10.2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase inEscherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.

Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry.

A technique using alkaline phosphatase histochemistry on routine sections of four jejunal biopsy specimens and one necropsy sample was applied to show that alkaline phosphatase activity, normally present in the brush border, occurs in the enterocytes of patients with microvillus inclusion disease. Sections were cut at 5 micron, mounted on to glass slides, and dried overnight at 37 degrees C before staining for alkaline phosphatase activity by the indoxyl phosphatase nitro blue tetrazolium method. Incubation periods amounted to 10 minutes for biopsy specimens and 30 minutes to one hour for necropsy samples. The demonstration of alkaline phosphatase activity in routinely processed biopsy specimens provides an effective, quick, and definitive test in the diagnosis of microvillus inclusion disease without recourse to electron microscopy.

Paper sections of macroenzymatically delineated myocardial necrosis

Until now, the disadvantages of the nitroblue tetrazolium method of detecting myocardial necrosis have been that the colour reaction tended to fade and the wet heart slices were inconvenient for demonstration and storage. We have therefore modified the rapid technique of mounting sections of whole organs on paper to preserve the colour reaction. We can now show the extent of necrosis in permanent, readily portable, paper-mounted heart sections within 24 hours (although 48 hours is more convenient). Multiple preparations from multiple slices can be prepared for clinicopathological correlation, comparison with other cases, teaching, presentation at meetings, use in the case notes and distribution with the postmortem report.

Influence of sulfinpyrazone on infarct size, hemodynamics, and arrhythmia following coronary occlusion in the pig.

We compared infarct size (nitroblue tetrazolium method), hemodynamic variables, and arrhythmias of a group of seven sulfinpyrazone-treated pigs (30 mg/kg/day for 7 days preoperatively, 30 mg/kg/i.v. 30 min before coronary ligation) with a control group of seven animals. Infarct size after ligation of the left anterior descending coronary artery was 20.3 +/- 1.1% in the sulfinpyrazone group and 20.3 +/- 0.5% in the control group. Heart rate, arterial blood pressure, cardiac output, and stroke volume were comparable in both groups before and for 60 min following ligation. There was no significant difference between the two groups in the number of premature ventricular contractions the PVC grade (Lown) or in the number of episodes of ventricular tachycardia or ventricular fibrillation during the 30-min observation period. We conclude that short-term administration of sulfinpyrazone has no effect on infarct size or arrhythmias in the acutely ischemic myocardium of the pig.

Reduction of myocardial infarct size by trapidil in anesthetized dogs.

We studied the effect of trapidil on acute experimental myocardial infarction in anesthetized, openchest dogs. The size of myocardial infarction 8 h after ligation of the left anterior descending coronary artery was determined by planimetry of myocardial slices stained by the nitroblue tetrazolium method. Systemic hemodynamic variables, epicardial ST-segment elevation, activity of serum creatine phosphokinase (CPK), and changes of myocardial blood flow in the ischemic area were measured. Infusion of 3 mg/kg trapidil reduced the size of infarction and ameliorated the infarction-induced deterioration of systemic hemodynamic variables, such as the decrease in left ventricular dP/dt, aortic blood flow, and regional endomyocardial blood flow in the ischemic area. This dose of trapidil also suppressed ST elevation and significantly inhibited the increase in activity of serum CPK. Hyaluronidase also reduced the size of infarction significantly. These results suggest that trapidil alters the course of acute myocardial infarction favorably, presumably by increasing regional endomyocardial blood flow.

Generation of hydrogen peroxide and superoxide anion from active metabolites of naphthylamines and aminoazo dyes: its possible role in carcinogenesis.

Autoxidation of active metabolites of naphthylamine and aminoazo dyes in neutral buffer generated hydrogen peroxide (H2O2) and superoxide anion (O-.2), as detected by the titanium sulfate method and nitro blue tetrazolium method, respectively. 2-Amino-1-naphthol, 1-amino-2-naphthol, 1-amino-4-naphthol, N-hydroxy-2-aminonaphthalene, N-hydroxy-1-aminonaphthalene, N-hydroxy-4-aminoazobenzene and N-hydroxy-4-methylaminoazobenzene generated H2O2 and O-.2, whereas 1-nitrosonaphthalene, 2-nitrosonaphthalene, 1-naphtylamine, 2-naphthylamine and non-carcinogenic aminonaphthols and naphthols generated no active oxygens. Catalase and superoxide dismutase were used to identify the formation of these active oxygens. For all compounds tested except nitrosonaphthalenes, good parallelism was found between the active oxygen formation and convertibility to free radicals. These results suggest a possible role of free radicals and subsequently formed active oxygens in aromatic amine carcinogenesis.

Effects of Anticoagulants and Storage on Granulocyte Function in Bank Blood

Abstract Storage of granulocytes for transfusion has not been practical because it has been considered that granulocyte function in bank blood is retained for only a few hours after collection. In the present study, granulocyte function was evaluated using the bactericidal assay and the quantitative nitroblue tetrazolium (NBT) method. Granulocytes from whole blood collected into acid citrate dextrose (ACD), citrate phosphate dextrose (CPD), heparin, ion exchange, and sodium citrate anticoagulants showed no functional impairment after 24 hr of storage at 4°C. With further storage, all granulocytes showed a loss of NBT activity. However, after 48 and 72 hr, granulocytes from whole blood stored in ACD and CPD killed the expected number of bacteria in the bactericidal assay. Thus, when tested in vitro, granulocytes maintain normal function, at least during the first 24 hr after collection when stored in certain anticoagulants under standard blood bank conditions.

Histochemical detection of three dehydrogenase systems in ameloblastoma

Three dehydrogenase systems—succinic dehydrogenase, DPN-dependent dehydrogenase (lactic, malic, α-glycerophosphate, and β-hydroxybutyric), and TPN-dependent dehydrogenase (isocitric, glucose-6-phosphate, and aconitase)—as well as monoamine oxidase, were histochemically studied in fifteen cases of ameloblastoma by the nitro blue tetrazolium method. All of the oxidative enzymes studied were confined to the neoplastic epithelium, but some of them were found in the stromal elements. The highest staining of the enzymatic activities was found in lactic dehydrogenase. The distribution patterns of several oxidative enzymes in ameloblastomas were similar to those of epidermoid carcinoma, especially in the case of the acanthoma type.