Prestaining Oxidation by Acidified H2O2 for Revealing Schiff-Positive Sites in Epon-Embedded Sections

Reliable production and identification of Schiff-positive sites on glutaraldehyde-osmium fixed 0.5-1 μsm Epon sections is accomplished by preoxidation of sections with 10% H₂O₂ acidified with H₂SO₄ (HPSA) to pH 3.2 (Pool, C. R., Stain Techn., 44: 75-9, 1969). Light green as a counterstain is used. Steps in the procedure are: HPSA, 1-2 min at 25-30 C; washing; 1% light green 3-5 min; brief rinse; Schiff reagent 1-3 min; washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the common occurrence of Schiff background staining in glutaraldehyde-fixed tissues and permits optimum penetration of staining solutions. Sections were attached to glass slides without adhesive and were processed in Coplin jars. Prior to drying, excess solutions should be drained and wiped away with lens tissue to prevent formation of precipitate on the sections.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

Oxidation of proteinaceous cysteine residues by dopamine-derived H2O2 in PC12 cells.

Cellular metabolism of dopamine (DA) generates H2O2, which is further reduced to hydroxyl radicals in the presence of iron. Cellular damage inflicted by DA-derived hydroxyl radicals is thought to contribute to Parkinson's disease. We have previously developed procedures for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues. Using these procedures, we identified ERP72 and ERP60, two members of the protein disulfide isomerase family, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, phospholipase C-gamma1, and thioredoxin reductase as the targets of DA-derived H2O2. Experiments with purified enzymes identified the essential Cys residues of creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, that are specifically oxidized by H2O2. Although the identified proteins represent only a fraction of the targets of DA-derived H2O2, functional impairment of these proteins has previously been associated with cell death. The oxidation of proteins that contain reactive Cys residues by DA-derived H2O2 is therefore proposed both to be largely responsible for DA-induced apoptosis in neuronal cells and to play an important role in the pathogenesis of Parkinson's disease.     

A Simple, Rapid Staining Procedure for Methacrylate Embedded Tissue Sections Using Chromotrope 2R and Methylene Blue

Sections of tissue embedded in glycol methacrylate can be stained in rapid sequence with solutions of 1% aqueous chromotrope 2R adjusted to pH 3 and 0.1% methylene blue to produce sufficient contrast and cellular detail to permit quick visual inspection and/or photomicrography. Solutions of these stains are simple to prepare and are stable over long periods. Staining of sections may be accomplished within six minutes.     

Staining of Different Endocrine Cells with Hydrochloric Acid-Toluidine Blue in Epon Embedded Rat Tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 turn 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

Oxidation of NADH by vanadium: kinetics, effects of ligands and role of H2O2 or O2.

The mechanism of oxidation of NADH by either vanadium(V) or vanadium(IV) was examined in the presence of reducing agents, complexing agents, and hydrogen peroxide. Reducing agents that stimulate the oxidation of NADH by V(V) include: a variety of cysteine analogues, glutathione, beta-mercaptoethanol, dithiothreitol, and ascorbate. Complexing agents which stimulate NADH oxidation by V(V) include cystine, glutathione disulfide, and dehydroascorbate. Vanadium(IV)-dependent systems which oxidize NADH include combinations of V(IV) with cysteine or air alone. Combination of either V(V) or V(IV) with hydrogen peroxide leads to NADH oxidation. Based on kinetic analysis and the use of the diagnostic inhibitors--superoxide dismutase, catalase, albumin, mannitol, ethanol, and anaerobic conditions--we have assigned two major mechanisms of NADH oxidation. One is the previously reported mechanism which involves V(V)-superoxide as the NADH oxidant. This reaction is inhibited by superoxide dismutase and anaerobic conditions but not by catalase or ethanol. This reaction is observed for V(V) in the presence of reducing agents and complexing agents. The second reaction mechanism operates when V(IV) comes in contact with hydrogen peroxide and involves V(III)-superoxide as the NADH oxidant. This reaction is inhibited by catalase (if unligated hydrogen peroxide is an intermediate) and superoxide dismutase but not anaerobic conditions or ethanol. This mechanism is observed for reactions of V(IV) with air or hydrogen peroxide.     

Pararosaniline Or Acriflavine-Schiff Staining of Epoxy Embedded Tissue After Periodic Acid Oxidation in Ethanol: A Method Suitable for Morphometric and Fluorometric Analysis of Glycogen

A method for preparing tissue sections for automatic image analysis of glyco-gen is described. Large semithin seaions of epoxy embedded tissue fixed in glutaraldehyde osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydra-tion before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

2H magnetic relaxation of 2H2O absorbed by wool.

D2O absorbed by intact wool fibers was studied by solid-state 2H nuclear magnetic resonance (NMR) spectroscopy. In wool fibers swollen in D2O, the deuteron transverse magnetization and the spin-locked magnetization revealed a non-exponential decay. At least two NMR phases with different sets of the NMR relaxation parameters, T(1rho) (2H) and T2 2H, have been detected that may be a manifestation of two different morphological phases of the cortex of the fiber.     

微胶囊固定化过氧化氢酶的制取及对H_2O_2的分解作用

以乙基纤维素为膜材,用溶液干燥法制取了微胶囊固定化过氧化氢酶,探讨了制取过程中明胶的加入对微胶囊产率及固定化过氧化氢酶活性的影响,同时论述了存放时间、温度以及环境ｐＨ值对微胶囊固定化过氧化氢酶稳定性的影响．深入研究了微胶囊固定化过氧化氢酶对Ｈ２Ｏ２的分解作用,获得了十分有意义的结果     

Histochemical Demonstration and Localization of H2O2 in Organs of Higher Plants by Tissue Printing on Nitrocellulose Paper

A sensitive tissue-print assay for the detection and histological localization of H2O2 in freshly cut organ sections was developed by impregnating nitrocellulose paper with a mixture of Kl and soluble starch. H2O2 transferred from the cut surface of the section to the dried paper forms I2, which can be visualized by the intensely colored I2-starch complex. The detection limit of the assay is in the range of 0.1 to 0.2 mmol L-1 H2O2. Due to the rapid immobilization of H2O2 in the paper, very clear prints of the tissue distribution of H2O2 can be obtained with a spatial resolution on the level of single cells. The application of this rapid and simple assay is explored in five experimental examples demonstrating that the in vivo level of H2O2 varies strikingly in different tissues and can be regulated by developmental factors such as hormones, light, and wounding. The results show that: (a) In the hypocotyl of soybean (Glycine max L.) seedlings the apoplastic H2O2 level increases strongly from top to base, accompanied by characteristic changes in its histological distribution. (b) In the epicotyl of pea (Pisum sativum L.) seedlings the induction of lateral expansion by ethylene is correlated with a depletion of H2O2 in the cell walls of the expanding tissues. (c) In the hypocotyl of bean (Phaseolus vulgaris L.) seedlings H2O2 is primarily localized in a ring of parenchymatic tissue between xylem and cortex next to lignifying cells but not in the lignifying cells themselves. (d) In the hypocotyl of sunflower (Helianthus annuus L.) and cucumber (Cucumis sativus L.) seedlings the light-mediated inhibition of elongation growth is correlated with a strong increase in H2O2 in the epidermis and in the vascular bundles. (e) Potato (Solanum tuberosum L.) tubers show high levels of H2O2 only in the outer cell layers but are able to accumulate H2O2 in the inner tissue upon wounding.