Staining of DNA with aqueous solution of basic fuchsin after phosphoric acid extraction of RNA.
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Biomedical subjects
Publications and source records attributed to M K Dutt.
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The use of the basic dye, Dahlia, which belongs to triphenylmethane group but without a primary amino group in its molecule has been described as useful in the staining of aldehyde groups of acid hydrolysed DNA in tissue sections following the conventional Feulgen procedure. Dahlia-SO2 prepared with sodium hydrosulphite is highly suitable when used at pH 4-0 to 5-0. The absorption characteristics of the stained nuclei indicate on the peak of maximum absorption at 560 nm, whereas, that of the aqueous dye solution is at 590 nm.
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The paper is a review of studies on Feulgen staining of nuclear DNA. For convenience, the paper is split into several sections, such as (a) the characteristic of the dyes used for the manufacture of Schiff reagent (b) preparations of Schiff reagent (c) fixation of the material (d) hydrolysis (e) staining with Schiff reagent (f) Schiff-type dyes and (g) use of Schiff-type dyes in quantitative cytochemistry. Barring the section on preparation of Schiff reagent that includes the classical methods, all other sections contain a comprehenside account showing progress made during the last 15 years. Under each section interpretations of new findings as far as possible, and in the section on staining with Schiff reagent, new approaches to its study have been presented. The section on Schiff-type dyes includes dyes with and without primary amino group.
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In the present study rat liver pieces fixed in 1) 10 per cent buffered neutral formalin, 2) 4 per cent glutaraldehyde, 3) Heidenhain's-Susa fixative and 4) Flemming's fluid, and following hydrolysis in 1-0 N HC1 at 60degreesC for varying time periods have been stained with the UV Feulgen procedure. The results of this study reveal that following hydrolysis for different time periods the tissue material fixed in formalin show the same staining pattern as those fixed in glutaraldehyde. The material fixed in Heidenhain's-Susa displays an intense Feulgen staining after two different times of hydrolysis, and that fixed in Flemming's fluid shows particular staining intensity for a prolonged time period thus indicating better preservation of DNA than in the materials fixed in the other three fixtatives.
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The paper embodies results of the use of 51 synthetic dyes, belonging to different chemical groups for staining of animal chromosomes following iodine-dye procedure. It has been found that some of these dyes can replace gentian violet, crystal violet and safranin when used after this procedure. It has further been found that the fluorescent dyes, acriflavine and acridine yellow can also be used to stain animal chromosomes and that some of the dyes belonging to one chemical group can be successfully used whereas others of the same group are of no use. Dyes of the monoazo group are absolutely useless. Amongst the dyes successfully used, the preparations remain stable when stained with most of them except methyl green, malachite green, brillant green, iodine green and cresyl violet and amongst acid dyes, acid fuchsin. Cytochemical studies presented herein indicate that the components of the animal chromosomes stainable with crystal violet are the nucleic acids and that these substances should be highly polymerised and should not be even in a semi-degraded state. Removal of any one of these nucleic acids makes the chromosomes unstainable with iodine-crystal violet.
In the experiments sections of rat tissues fixed in 10 per cent buffered neutral formalin for 3 hour and treated for 15 minutes with different chemical reagents such as pyridine, tributylamine, urea, tris sodium nitrite and sodium hydroxide were subjected to hydrolysis in 6 N HCl at 25 degrees C for 20 minutes and stained by the UV Feulgen technique. The results reveal a far more intense staining of the nuclei in tissues treated with any of the chemicals mentioned than in untreated controls. The possible role of these chemicals in enhancing the staining intensity is discussed.
Sections of rat liver fixed in CRAF III and Nawaschin's fixative in Dutt's modification were subjected to hydrolysis in 1N HCl at 60 degrees C for different periods of time and to Schiff's staining according to the UV Feulgen technique. The study showed that Feulgen reaction intensity depends upon time of hydrolysis, optimum coloration being possible only after 10-15 min of hydrolysis. Prolongation of hydrolysis beyond this time produced decreased staining intensity which is retained for further 35 min of hydrolysis thus forming a plateau. Further prolongation of hydrolysis results in gradual deterioration of the staining intensity which culminates in utterly pale coloration of the nuclei after one hour's hydrolysis. A possible explanation for this phenomena is suggested.
The paper describes a highly satisfactory method for in situ localization of DNA in tissues fixed in picro-formol-acetic acid or picro-formol-acetic-chromic acid mixtures following a technique in the Feulgen procedure as devised by the author. Mammalian tissues fixed in these fixatives can be hydrolysed in 6N HCl at 35 degrees C for 10 min, rinsed in water, stained with Schiff reagent after exposing the sections under UV light for 10 min, washed in water, dehydrated through a graduated series of ethanol, cleared in xylol and mounted in DPX. Sections of tissues fixed in picro-formol-acetic-chromic acid mixtures after acid hydrolysis when stained with an aqueous solution of basic fuchsin are also found to be very satisfactory for in situ localization of DNA.