Methyl Green-Pyronin staining with pure Pyronin Y.
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Methyl Green↗
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Nucleic acid staining with the methyl green-pyronin method. A comparison of the use of pure dyes and commercially available dyes.
We compared the staining obtained using commercially available pyronin Y samples with that obtained using pure pyronin Y in a standardized methyl green-pyronin procedure. In addition, the importance of the dye content of the anhydrous dye was investigated by varying the dye content by the addition of pure pyronin Y to one of the commercially available pyronin Y samples. We found that, for routine histological work, commercially available pyronin Y samples may produce acceptable results provided the sample can be shown by spectrophotometry to contain at least 43% pyronin Y.
Animals↗
An investigation of new commercial samples of methyl green and pyronin Y.
New commercial samples of Methyl Green (Gurr Certistain), Pyronine G (Gurr Certistain) and Pyronin Y (Polysciences) have been investigated using spectrophotometry, thin layer chromatography and nuclear magnetic resonance, in addition to standardized simultaneous and sequential staining methods using purified Ethyl Green and pure Pyronin Y as reference dyes. The Methyl Green was found to be Ethyl Green contaminated with Crystal Violet. It did not have any advantages compared with Ethyl Green supplied by American dye companies. The Pyronine G sample was Pyronin Y with a high dye content that gave good staining results when used with purified Ethyl Green. Pyronin Y from Polysciences was found to be essentially pure Pyronin Y.
Chromatography, Thin Layer↗
Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.
For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA. The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen reaction. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm. After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum. We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.
Animals↗
Purity of commercial non-certified European samples of Pyronin Y.
The purity of six European non-certified samples of Pyronin Y was compared with that of two American samples certified by the Biological Stain Commission. The methods used were spectrophotometry and a Methyl Green-Pyronin staining test (both as applied by the Biological Stain Commission), thin layer chromatography, mass spectrometry, determination of pH, and content of some electrolytes. It was found that none of the European batches of Pyronin Y passed the complete test as prescribed by the Biological Stain Commission. Their dye content was uniformly low (between 5 and 19%). Furthermore, thin layer chromatography and mass spectrometry revealed that two of the dye samples contained no Pyronin Y or only traces. It is concluded that assessment of an unknown sample of a dye labelled Pyronin Y should be initiated with thin layer chromatography. The pH and content of electrolytes in an aqueous solution of the dye should also be determined in order to obtain reproducible staining results. Finally, the value of the work performed by the Biological Stain Commission is underlined, although more sophisticated methods are necessary for testing the purity of dyestuffs.
Cations↗
Standardized methyl green-pyronin Y procedures using pure dyes.
Fully standardized Methyl Green-Pyronin methods are presented. Pure Pyronin Y and purified Methyl Green or Ethyl Green are used either simultaneously in one dye bath or are used as a sequence of Pyronin Y and Ethyl or Methyl Green. Both methods, as shown by enzymatic pretreatment, give a reliable and reproducible staining in DNA with Ethyl or Methyl Green and of RNA with Pyronin Y on Carnoy fixed material. On formaldehyde fixed material it was found advantageous to use the sequential method as chromatin was hereby stained green instead of blue as seen with the simultaneous method.
Animals↗
The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.
Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.
Animals↗
[Concentration and temperature effects in interactions of the dye pyronine G with polynucleotides].
The interaction of poly(A) and poly(A).poly(U) with pyronine G dye depending on the concentration of components and temperature was studied spectrophotometrically in the visible and UV ranges at pH (6.86). It was found that the interaction of pyronine G with poly(A) and poly(A).poly(U) results in the formation of two types of complexes. The relation of the equilibrium concentrations of these complexes depends on the initial concentrations of the components in solution. The formation of complex I results in shifting the spectrum towards the short wave range with regard to the monomer band and reflects the aggregation of the dye cations. Complex II is characterized by the shift towards the long wave range. Complex II is formed in considerable amounts for poly(A).pyronine G system at large P/D and for poly(A).poly(U).pyronine G system at P/D = 5-6 and is probably due to the interaction between the dye and polynucleotides of the intercalation type or reflects the interaction between the dye and two negatively charged phosphate groups. Analysis of temperature measurements of spectra confirms the formation of various types of complexes in the system studied.
In Vitro Techniques↗
[Spectroscopic evidence of the interaction of the dye pyronine G with polynucleotides].
The interaction of pyronine G with mono- (AMP) and polynucleotides, i. e. poly(A) and poly(U), was studied at different pH values and temperatures, both in the visible and near UV ranges. It was found that pyronine G interacts only with purine type polynucleotides at neutral and alkaline pH. This was confirmed by the appearance of a new dye absorption band in the visible range and the existence of a hypochromic effect in the UV. The spectroscopical evidence for the formation of pyronine G--AMP or poly(U) complex has not been found at any conditions. With these results in view, possible binding mechanism of pyronine G to polynucleotides are discussed.
Adenosine Monophosphate↗
The effects of pyronin on sprouting and regeneration of mouse motor nerves.
Administration to mice of a 0.1% solution of pyronin G in their drinking water caused an acceleration both of axonal sprouting from nodes of Ranvier in partly denervated gluteus maximus muscles, and of motor nerve regeneration following a crush to the soleus nerve. Sprouting from soleus motor nerve terminals in response to botulinum toxin-induced paralysis was, however, unaffected. Removal of degenerating axons following nerve section was also accelerated by pyronin treatment. Pyronin is therefore likely to act upon the process of Wallerian degeneration, rather than upon intact motor nerves directly.
Acetylcholine↗
Chromosomal aberrations and SCEs in Allium cepa root-tip cells treated with caffeine and pyronin Y.
The effectiveness of caffeine and pyronin Y in the induction of both chromosomal aberrations and sister-chromatid exchanges (SCEs) in root meristematic cells of A. cepa was studied. The rate of SCEs proved to be increased when 5-bromo-2'-deoxyuridine- (BrdU) substituted chromosomes were allowed to replicate in thymidine (dT) for a second S period simultaneously with caffeine or pyronin Y. In contrast, only caffeine was able to induce aberrations in BrdU-substituted chromosomes, while pyronin Y seemed to be ineffective at the doses employed.
Bromodeoxyuridine↗
1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems.
We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.
Animals↗
Pyronin--a fluorescent indicator of membrane potential of the mitochondria. Mechanism of action.
The effect of the functional state of isolated mitochondria on the fluorescence of pyronin added to the suspension is studied. It is shown experimentally that the intensity of fluorescence changes parallel to the degree of energization of the mitochondria. The magnitude and the sign of the fluorescent changes depend on the pyronin/mitochondria ratio and the length of the wave of excitating light. Model experiments show that the effects observed are probably due to the electrophoretic redistribution of pyronin between the medium, the mitochondrial membrane and the matrix, induced by the membrane potential of the mitochondria. It is assumed that pyronin acts as vector fluorescent indicator, similar to anilinonapthalenesulphonate.
Animals↗
Orthochromatic and metachromatic staining reactions by pyronin Y on Epon semithin sections.
Semithin sections from glutaraldehyde-fixed, Epon-embedded tissues were stained by aqueous solutions of pyronin Y at increasing concentrations (from 10(-6) to 10(-3) mol). Mucopolysaccharide containing structures (e.g. mucin) were found stained in orange, meanwhile the chromatin and remaining tissue components appeared in a bright pink-red color. Cytophotometric measurements showed that a metachromatic shift occurs in the mucin content from goblet cells after pyronin Y staining at 10(-3) mol. Some features of the metachromatic reactions by cationic dyes are briefly discussed.
Animals↗
Pyronin-phosphotungstic acid: spectral characteristics and staining pattern on Epon semithin sections.
Phosphotungstic acid (PTA) binds to the basic dye pyronin Y to give a non fluorescent complex with absorption characteristics which are different from those of the free dye. The use of this complex on different cells and Epon embedded tissues revealed no staining affinity and only certain hydrated tissue components as starch showed a light reaction with the pyronin-PTA. The role played by the hydratation degree of certain components from epoxy-embedded tissues on the reaction mechanisms observed with some dyes and electron contrasting agents is briefly discussed.
Animals↗
Kinetic determination of tellurium based on its inhibitory effect on the palladium(II)-catalysed reaction between pyronine G and hypophosphite ion.
A kinetic method for the determination of Te based on its inhibitory effect on the PdII-catalysed reaction between pyronine G and H2PO2- is described. The influence of experimental variables on the rate of the process and the potential interfering effect of a large number of ions has been studied. Under the selected experimental conditions: 6 x 10(-5) M pyronine G; 0.6 M H2PO2-; pH 2.6, adjusted with Britton-Robinson buffer; 0.80 microgram ml-1 of PdII; and a temperature of 22 +/- 0.2 degrees C, Te was determined in the concentration range 0.08-0.85 microgram ml-1. The method was applied to the determination of Te in waters and lead concentrates.
Carbonated Beverages↗
Iron pyronine and alcian blue for staining acid mucin in plastic-embedded sections.
Iron pyronine Y and Alcian blue demonstrated sulfated and nonsulfated acid mucin, respectively, in plastic-embedded sections. Safran used in combination with these two dyes stained collagen and some reticulum fibers. Sulfated acid mucin was red, while nonsulfated acid mucin stained blue; collagen appeared yellow to greenish yellow. The iron pyronine Y-Alcian blue-safran staining method, when used as in the present article, produces excellent cellular visualization of sulfated and nonsulfated acid mucin.
Alcian Blue↗
Methyl green-pyronin with hematoxylin and orange G for the identification of inflammatory cells in tissue sections.
Methyl green-pyronin is a notoriously difficult stain to reproduce. Although very useful in detecting cells containing substantial amounts of RNA, it is of limited use in broader problems of cell identification. By careful standardization of the proportions of methyl green to pyronin and combination of these stains with hematoxylin to enhance nuclear contrast and with orange G to improve connective tissue staining, it was possible to produce a consistently reliable staining preparation in which it is possible to identify all the component cells of a mixed inflammatory infiltrate in routine paraffin sections.
Animals↗