Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

A Mixture of Methyl Green and Pyronin for Cell Fractionation Studies

A staining mixture consisting of 0.57% methyl green and 0.1 1 % pyronin B (calculated from the actual dye content) dis- solved in glycerol, 20 ml.; 2% aqueous phenol, 100 ml.; and 95% ethanol, 25 ml., was found to be optimum for differentiating cell components containing desoxypentose and pentose nucleic acids. The stain can be used for either fresh suspensions or unfixed dried smears of tissue homogenates. Nuclei are stained bright blue, and nucleoli and cytoplasmic particles, bright pink.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

Effects of Temperature, Poststaining Rinses and Ethanol-Butanol Dehydrating Mixtures on Methyl Green-Pyronin Staining

Methyl green GA (Chroma) and pyronin GS (Chroma) were used. Procedure recommended: Stain for 1 hr at 37 C in a purified 0.5% aqueous methyl green, buffered to pH 4.1 with Walpoles acetate buffer, and containing 0.2% pyronin; rinse for 1-2 sec in ice-cold distilled water; blot sections evenly, and rinse with vigorous agitation in t-butanol; dehydrate in 2 changes of t-butanol for 5 min each; clear in xylene and mount. This technique results in a consistent staining pattern for qualitative nucleic acid differentiation, whereas older methods have been only partly satisfactory. Rinsing in ice-cold water is a critical step; t-butanol was superior to n-butanol and to ethanol-butanol mixtures for dehydration. Staining at 25-27 C is feasible hut less effective.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

A Six-Dye Staining Schedule for Sections of Mesquite and Other Desert Plants

Materials are fixed in FPA (formalin, 2; propionic acid, 1; 70% ethanol, 17). Paraffin sections on slides are brought to 50% ethanol and stained as follows: (1) in Bismarck brown Y, a 0.02% solution in 0.1% aqueous phenol, 10-30 min; wash 30 sec in 0.7% acetic acid, and wash in distilled water 20-30 sec; (2) in crystal violet, 1% in 70% ethanol alkalinized with 1 drop of 1 N NaOH per 100 ml, 12-35 min; wash 30-60 sec in tap water to remove excess stain, and rinse 0.5 sec in 70% ethanol; then mordant in I₂-KI, 1% each in 70% ethanol, 40 sec, and rinse in 70% ethanol 2-5 sec; (3) in a mixture containing 0.4% acid fuchsin and 0.6% crythrosin B in 70% ethanol about 0.5 sec; rinse in 70% ethanol 5-15 sec to remove excess red; dehydrate in 70%, 95%, and absolute ethanol, 2-3 sec each; (4) in fast green FCF, 0.5% in a mixture of equal parts of methyl cellosolve, absolute ethanol, and clove oil, 5-15 sec; rinse in a mixture of clove oil, 10 ml; absolute ethanol, 100 ml; and methyl cellosolve, 10 ml, 5-7 sec; (5) in orange G, 0.75 gm in a mixture of clove oil, 40 ml; absolute ethanol, 40 ml; and methyl cellosolve, 60 ml, 5-30 sec; rinse clean in a 1:1 mixture of xylene and absolute ethanol, 5-20 sec Complete the clearing in pure xylene, 3 changes, 1.5 min in each, and apply a cover glass with synthetic resin. Slides are agitated in all steps except Bismark brown Y, crystal violet, and the xylenes. Contrast and staining intensity are adjusted by varying staining times in the dye solutions.     

Qualitative Analysis by Thin-Layer Chromatography of Some Common Dyes Used in Biological Staining

Thin-layer chromatography will resolve impurities in commercial dyes, and will do so much faster than paper chromatography. Solvent systems consisting of (a) n-propanol: n-butanol: NH₄OH (conc.): H₂O—4:4:1:1; (b) n-propanol: NH₄OH (conc.): H₂O—8:1:1 on silica gel G plates; and (c) n-propanol: NH₄OH (conc.): H₂O-7:2:1 on Adsorbosil plates were found to be the most effective. Dyes studied were azure A, azure B, azure C, methylene blue, toluidine blue O, thionin, pyronin B, pyronin Y, methyl green, crystal violet amido black 10B and buffalo black (NBR).     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time,dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Methyl Green-Pyronin for Staining Autoradiographs of Hydroxyethyl Methacrylate-Embedded Lymphoid Tissue

Cells in the spleen in DNA-synthesis were labelled with tritiated thymidine. Tissue was fixed for 12 hr in 10% neutral formalin, washed for 4 hr in tap water and dehydrated through 70% and absolute ethanol. The tissue blocks were infiltrated overnight with a mixture consisting of glycol methacrylate, 80 ml; polyethylene glycol 400, 12 ml; and benzoyl peroxide, 0.27 gm. Specimens were cast in BEEM capsules with the final embedding medium consisting of 42 parts of the infiltration medium and 1 part of an acceleration mixture. This mixture consisted of N,N-dimethylaniline, 1 part and polyethylene glycol 400, 15 parts. The blocks hardened in 30 min and were sectioned with an ultramicrotome fitted with glass knives. Sections were coated with Ilford K5 liquid emulsion and exposed for 2 wk. Methyl green-pyronin staining of autoradiographs was carried out at pH 4.1 in acetate buffer containing 0.5% methyl green (Allied Chemicals) and 0.2% pyronin GS (Chroma). Staining was for 30-60 min, after which sections were washed for 1 min in water, blotted, allowed to dry, and mounted in Canada balsm. The procedure resulted in good quality autoradiographs in which the degree of basophilia of labelled cells could be assessed.     

Counterstaining Golgi-Cox Impregnations with Luxol Fast Blue as a Myelin Stain

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K₂Cr₂O₇, 1 gm; HgCl₂, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K₂CrO₄, 0.8 gm; Na₂WO₄, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO₃, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5-10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5-60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li₂CO₃ for 3-4 min. Differentiate in 70% ethanol and place in water. Immerse for 5-15 min in a mixture of two solutions: A. cresylechtviolet (Otto C. Watzka, Montreal), 2 gm; 1 M acetic acid, 185 ml; B. 1 M sodium acetate, 15 ml; distilled water, 400 ml; absolute ethanol, 200 ml. Dehydrate to 3:1 ethanol-chloroform. Clear in toluene and apply a coverslip. The technique produces fast Golgi-Cox impregnated neurons against a background of counterstained myelinated fibers. Patterns of the myelinated fibers can be used to localize impregnated neurons.     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Spectrophotometry of Dyes: 1. Methyl Green. 2. Pyronin

Comparisons of absorption peaks of seven samples of methyl green showed that two different types of the dye were represented. One type (2 samples) had the visible peak near 617 mμ; the other (4 samples) near 630 mμ, while one sample was intermediate in spectral characteristics. Using these findings as a means of differentiating between heptamethyl and hexamethylethyl pararosanil-in is suggested. The Y and B forms of pyronin were found to be readily distinguishable by comparing their absorption maxima (Y, 546 mμ, B, 557-8 mμ). A check on the application of Beer's law of dilution showed that it held (1-3 mg./liter) for pyronin and that the relative effect of dilution was a slow increase with pyronin but a rapid decrease with methyl green.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

Differential Staining of Mucin Granules from Epoxy Resin Sections by a Phosphotungstic Acid-Methyl Green Procedure

After treatment of epoxy resin semithin sections from glutaraldehyde fixed rat large intestine with 5% aqueous phosphotungstic acid (PTA), staining with unpurified 0.2% solutions of methyl green at 60 C for 5 min produces a color differentiation between mucin granules of goblet cells. Some mucin granules and the glycocalyx appear deep green while the remaining granules, luminal mucin and collagen fibers are pink. The known contamination of unpurified methyl green with crystal violet seems to be responsible for the pink staining reaction of the latter structures, which also present an orange-red fluorescence under green exciting light. Electron microscopic observations show selective contrast of mucin granules which appear with a different amount of PTA deposits. This procedure is useful to reveal the heterogeneity of mucin granules in light and electron microscopy.     

Chlorazol Black E as a Stain for Tension Wood

Sections containing gelatinous fibers were cut at 15 μ from material both fixed and stored in formalin-acetic-alcohol, 5:5:90 (of 70%). These sections were stained 5 min in a 1% aqueous solution of lignin pink (G. T. Gurr), differentiated quickly in water, soaked 5 min in 95% ethyl alcohol, dehydrated in absolute ethyl alcohol and counter stained 5 min with a 1% solution of chlorazol black E (G. T. Gurr) in methyl cellosolve, followed by dehydration in absolute ethyl alcohol, clearing in xylene and mounting in Canada balsam. The gelatinous layer was sharply defined as a dense black zone whilst the remainder of the cell wall stained light pink. The specificity of the technique was superior to that of safranin and light green, and was not easily obscured by overstaining. The technique is particularly useful for locating small zones of gelatinous fibres, and for photomicrographical work.     

Red-blue staining of hydrolysed nucleic acids in paraffin sections

A previous treatment with 10% HC1 in tetrahydrofuran for 2-3 min at 37° C hydrolyses DNA while substantially preserving RNA in formol-fixed paraffin sections. If this treatment is followed by dyeing with basic fuchsin-thiazine or oxazine mixtures, the basic fuchsin stains DNA, the blue dye cytoplasmic RNA, though nucleolar RNA is not well preserved. A specimen sequence is to treat the hydrolysed section with a mixture of 1% aqueous trimethylthionin (Chroma), 15 ml; 0.1% basic fuchsin (G. T. Gurr), 4 ml; and glacial acetic acid, 1 ml. Stain for 15-30 min, dehydrate in acetone, then pass sections through xylene to polystyrene. The specificity of this stain for cytoplasmic RNA is sharper than that of methyl green-pyronin; hence the technic given can be a useful addition to the standard Unna-Pappenheim procedure.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.