scholarly journals NEW HISTOCHEMICAL TECHNIQUES FOR THE DEMONSTRATION OF TISSUE OXIDASE (CYTOCHROME OXIDASE)

1959 ◽  
Vol 7 (2) ◽  
pp. 112-122 ◽  
Author(s):  
M. S. BURSTONE
Keyword(s):  
Frozen Sections ◽  
Freezing And Thawing ◽  
Tissue Slices ◽  
Fresh Tissue ◽  
Open Chain ◽  
Cobalt Nickel ◽  
Glass Slides ◽  
Naphthoic Acid ◽  
Potential Applications ◽  
Cadmium Lead

A group of complex naphthols, as well as open chain and cyclic methylene compounds, were found to be useful new reagents for the demonstration of tissue oxidase in conjunction with N-phenyl-p-phenylenediamine. The methylene compounds constitute a new class of histochemical reagents. N-alkyl amines (e.g. N,N-dimethyl arylamines) are toxic to the enzyme and are to be avoided. Complex amines are inhibitory and are likewise not recommended. As with the original "Nadi" technique, the reaction was inhibited by cyanide, azide, and sulfide. The distribution of staining in tissues was essentially identical with all reagents. Although the reaction was augmented by cytochrome c, this expensive reagent is not neeeded with the present techniques. The indoaniline and azamethine dyes produced are highly insoluble and result in permanent or relatively permanent preparations. Most of the dyes are capable of complexing with mercury, molybdenum, cobalt, nickel, cadmium, lead, uranium, and iron, and thus may have potential applications in electron microscopy. Among the naphthols 1-hydroxy-2-naphthoic acid (IV), 1-hydroxy-2-acetonaphthone (V), and a derivative of 1-hydroxy-2-naphthanilide (IX) are very useful couplers. Useful methylene compounds are exemplified by 1-phenyl-3(m-nitrobenzamidopyrazolone) (XII), naphthol AS-LG (XVIII), and naphthol AS-L3G (XX). Use of methylene compounds results in highly stable incubating solutions. All substrates are commercially available and inexpensive. Unfixed frozen sections mounded on glass slides were used. Considerable caution must be exercised in attempts to localize oxidative enzyme activity at the cytologiocal level in unfixed frozen sections. This is due to the disruption of mitochondria and other cytological constituents which may occur during freezing and thawing. An alternative technique was evolved. This consisted of incubating fresh tissue slices, freezedrying them, followed by embedding in paraffin. Sections were then cut, dewaxed, and mounted. Preliminary results indicate that tissue oxidase may also be demonstrated using a mixture of amines (e.g., N-phenyl-p-phenylenediamine and 2-methoxy-N-phenylenediamine) which are capable of forming complex azine dyes. The possibility of demonstrating other enzymes of the oxidase dehydrogenase complex warrants further investigation.

scholarly journals Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling

Cancer ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 483-491 ◽  
Author(s):  
Wilfrido D. Mojica ◽  
Amy V. Rapkiewicz ◽  
Lance A. Liotta ◽  
Virginia Espina
Keyword(s):  
Molecular Profiling ◽  
Frozen Sections ◽  
Fresh Tissue

Effects of ageing and salicylate on beetroot mitochondria

2000 ◽  
Vol 27 (5) ◽  
pp. 445
Author(s):  
Felicity Johnson Potter ◽  
Ellen Bennett ◽  
Joseph T. Wiskich
Keyword(s):  
Mitochondrial Respiration ◽  
Alternative Oxidase ◽  
Alternative Pathway ◽  
The Other ◽  
Tissue Slices ◽  
Fresh Tissue ◽  
Outer Membranes ◽  
Ageing Treatment ◽  
Specific Induction ◽  
Increased Activity

Root tissue slices of Beta vulgaris were aged by aerated mixing for 5 d in 0.1 mM CaSO4 solution with and without 0.5 mM salicylate (pH 7.0). Mitochondria, isolated from treated and fresh beetroot tissue, were investigated, and the activities of the NAD(P)H oxidising pathways of the mitochondrial inner and outer membranes, together with the activity of the alternative pathway, were measured. From these we determined which non-phosphorylating pathways of mitochondrial respiration were induced by the treatments. The presence of salicylate in the ageing treatment was shown to increase the activity of the alternative pathway as well as the level of alternative oxidase protein. This increase was above that seen in the ageing treatment alone. Investigation of the other non-phosphorylating pathways of mitochondrial respiration indicated that while they also increased during ageing, they were not specifically affected by salicylate treatment. The specific induction of the alternative pathway increased activity by almost 4-fold from the fresh tissue and 2-fold from the aged tissue rates.

scholarly journals ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

1967 ◽  
Vol 34 (3) ◽  
pp. 787-800 ◽  
Author(s):  
Arnold M. Seligman ◽  
Robert E. Plapinger ◽  
Hannah L. Wasserkrug ◽  
Chandicharan Deb ◽  
Jacob S. Hanker
Keyword(s):  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Tetrazolium Salt ◽  
Tissue Slices ◽  
Fresh Tissue ◽  
Fixed Tissue ◽  
Cytochrome Oxidase Activity ◽  
Renal Tubular Cells ◽  
Renal Tubular ◽  
Rat Tissue

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).

scholarly journals FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

1961 ◽  
Vol 9 (2) ◽  
pp. 415-427 ◽  
Author(s):  
Donald G. Walker ◽  
Arnold M. Seligman
Keyword(s):  
Divalent Cations ◽  
Succinic Dehydrogenase ◽  
Staining Procedure ◽  
Frozen Sections ◽  
Tissue Slices ◽  
Fixed Tissue ◽  
Cytochemical Demonstration ◽  
Hematoxylin Staining ◽  
Fresh Frozen ◽  
Duodenal Epithelium

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q10, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.

PRESSURE-ASSISTED FREEZING AND THAWING: PRINCIPLES AND POTENTIAL APPLICATIONS

2000 ◽  
Vol 16 (4) ◽  
pp. 453-483 ◽  
Author(s):  
J. C. CHEFTEL ◽  
J. LÉVY ◽  
E. DUMAY
Keyword(s):  
Freezing And Thawing ◽  
Potential Applications

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

1995 ◽  
Vol 1 (5) ◽  
pp. 217-230
Author(s):  
A. Kent Christensen ◽  
Terry B. Lowry
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light And Electron Microscopy ◽  
Ice Crystal ◽  
Freezing Damage ◽  
Frozen Sections ◽  
Glass Slides ◽  
Frozen Tissue ◽  
Crystal Damage ◽  
Ultrathin Frozen Sections

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about −117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at −155 to −170°C to produce semithin frozen sections (0.5 to 3 μm thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

scholarly journals Digital Histology by Phase Imaging Specific Biomarkers for Human Tumoral Tissues Discrimination

2021 ◽  
Vol 11 (13) ◽  
pp. 6142
Author(s):  
José Luis Ganoza-Quintana ◽  
Félix Fanjul-Vélez ◽  
José Luis Arce-Diego
Keyword(s):  
Fractal Dimension ◽  
Phase Contrast ◽  
Tissue Type ◽  
P Value ◽  
Phase Imaging ◽  
The Novel ◽  
Label Free ◽  
External Scale ◽  
Tissue Slices ◽  
Fresh Tissue

Histology is the diagnosis gold standard. Conventional biopsy presents artifacts, delays, or human bias. Digital histology includes automation and improved diagnosis. It digitalizes microscopic images of histological samples and analyzes similar parameters. The present approach proposes the novel use of phase contrast in clinical digital histology to improve diagnosis. The use of label-free fresh tissue slices prevents processing artifacts and reduces processing time. Phase contrast parameters are implemented and calculated: the external scale, the fractal dimension, the anisotropy factor, the scattering coefficient, and the refractive index variance. Images of healthy and tumoral samples of liver, colon, and kidney are employed. A total of 252 images with 10×, 20×, and 40× magnifications are measured. Discrimination significance between healthy and tumoral tissues is assessed statistically with ANOVA (p-value < 0.005). The analysis is made for each tissue type and for different magnifications. It shows a dependence on tissue type and image magnification. The p-value of the most significant parameters is below 10−5. Liver and colon tissues present a great overlap in significant phase contrast parameters. The 10× fractal dimension is significant for all tissue types under analysis. These results are promising for the use of phase contrast in digital histology clinical praxis.

Effects of pH, UV-B radiation and NaCl on anthocyanin stability from vivid blue petals of Clitoria ternatea L., a potential natural colourant from legume crop

2018 ◽  
Vol 47 (6) ◽  
pp. 507-510 ◽  
Author(s):  
Noraini Mahmad ◽  
R.M. Taha
Keyword(s):  
Spectrophotometric Analysis ◽  
Coating Technology ◽  
Clitoria Ternatea ◽  
Content Type ◽  
Food Dye ◽  
Legume Crop ◽  
Glass Slides ◽  
Potential Applications ◽  
Effects Of Ph ◽  
New Finding

Purpose The purpose of this study is to investigate the effects of pH, UV-B radiation and NaCl on anthocyanin extracted from vivid blue petals of Clitoria ternatea L. (legume crop), as a potential natural colourant for food, dye or coating technology. Design/methodology/approach The anthocyanin from petals of Clitoria ternatea was extracted using 0.5 per cent trifluroacetic (TFA) in methanol solution. The liquid colourant was exposed to different pH (1, 4.5 and 5.5), UV-B radiation and sodium chloride (NaCl). The results were compared using UV-vis spectrophotometric analysis. Findings Anthocyanins are sensitive and quickly degrade in the presence of light. In the dry powder form, the anthocyanin is easier to maintain and preserve (storage). Research limitations/implications Anthocyanins extracted from vivid blue petals of Clitoria ternatea L. are sensitive and quickly degrade in the presence of light. Practical implications The anthocyanin pigments extracted from Clitoria ternatea L. petals with methanolic acid were successfully coated on glass slides. The combination of binders and pigments had produced environmental paint which added with stabilisers (additives) for better durability. Acrylic has been known for its high weathering and embrittlement resistance, good mechanical and electrochemical properties and gloss retention. Social implications This anthocyanin is suitable as natural colourant especially in baby products, cosmetics production or for coating and varnish application. Originality/value Till date, the natural colourant of Clitoria ternatea L. petals is widely used in food. However, this result is a new finding, as there is no report on the potential of applications of this natural colourant for coating technology. Therefore, the current study with appropriate extraction method was significantly based on the relevant literatures of coating production from pigment by using other plant species. The findings and conclusion highlight the practicality as the potential applications in coating technology.

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