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.     

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.     

Purity of commercial non-certified European samples of Pyronin Y

Summary 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.     

Spectroscopic investigations on the binding of Pyronin Y to human serum albumin

The interaction of Pyronin Y with human serum albumin (HSA) has been investigated systematically by fluorescence, absorption, fluorescence decay lifetime measurements, FTIR, synchronous fluorescence spectroscopy, and molecular modeling method. The spectroscopic and fluorescence quenching experiments show that Pyronin Y may show a static quenching mechanism with HSA. The specific binding distance of 1.96 nm between HSA and Pyronin Y was obtained via Förster non-radiation energy transfer method. The thermodynamic parameters indicate that the electrostatic interactions play a significant role during the binding process. In addition, synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of HSA were not influenced with the addition of Pyronin Y. The obtained results can be of biological significance in photodynamic therapy.     

Newer Applications of the Histological Stain Prepared from Pterocarpus Santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn, has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Chemical Aspects of Santalin as a Histological Stain

Recent research on the chemical nature of the red dyes isolated from Pterocarpus santalinus and certain West African plants, viz., Baphia nitida, Pterocarpus osun and Pterocarpus soyauxii, have been reviewed. P. santalinus contains santalins A, B and C., but no santarubin. Santalins and santarubins have been found in P. osun, P. soyauxii and B. nitida. The structural formulae of the santalins are presented and their differences from santarubins indicated. Santalins A and B have some similarities in structure with hematein. This is probably responsible for their staining properties; the possible mechanism of staining is discussed.     

Schiff-Type Reagents in Cytochemistry. 2. Detection of Primary Amine Dye Impurities In Pyronin B and Pyronin Y(G)

Many batches of pyronin B (C.I. 45010), pyronin Y or G (C.I. 45005), and acridine red (C.I. 45000) produce positive Feulgen or PAS reactions when their 0.25% solutions are saturated with SO₂ and used on acid-hydrolyzed or periodate-oxidized tissue sections. These dyes behave as Schiff-type reagents and stain aldehyde-containing structures orange, brown, pink, red, or violet, depending on the particular batch used. The most frequent contaminants are violet and are nonfluorescent. Aldehyde groups are stained by these dyeSO₂ solutions as is shown by using unhydrolyzed controls in the Feulgen reaction and unoxidized controls in the PAS reaction, and by dye solutions lacking SO₂. Other procedures included reactions with aldehyde-blocking reagents, treatment with deoxyribonuclease and diastase, and extraction of nudeic acids with trichloroaeetic acid. The standard Schiff reagent was used in the Same procedures as a basis for comparing results. Since the Schiff-aldehyde reaction requires a dye with a primary amine group and since true pronins contain only secondary or tertiary amines, the positive histochemical results are evidently caused by dye contaminants possessing primary amine groups. The PAS reaction is more sensitive than the Feulgen reaction in detecting dye contaminants. Tissues used were chiefly formalin-fixed mouse intestine and ascites cells. Seventy-five commercial pyronins were studied from 21 different firms. Among 19 batches of pyronin B, 14 were found to contain primary amine dye contaminants. Among 39 batches of pyronin Y(G), 19 contained similar primary amine dye contaminants. Of the 8 batches of acridine red tested, 7 were found to contain primary amine dye contaminants. Nine commercial mixtures of methyl green-pyronin were studied and 4 were found to be likewise contaminated, but these reactive dye contaminants in them are apparently not associated with methyl green. A tabulated summary of the pyronin batches containing primary amine contaminants, and a list of sources and distributors of pyronin dyes are included.     

An investigation of new commercial samples of Methyl Green and Pyronin Y

Summary 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.     

Dye standards, Part II.1: Pyronin Y (CI 45005)

The names and affiliations of the SRMTS members are cited in Part I: terminology and general principles,Histochem. J. (1992)24, 217–19.     

The Use of Bismarck Brown Y in some new Stain Schedules

The staining quality of Bismarck brown Y may be improved and sterility maintained by adding 5% phenol to a 1% aqueous solution. Use the phenolic Bismarck brown in combination with iron alum hematoxylin except for stripped epidermis in the following procedures:

Stem and Root Schedule: Mordant sections from water in 4% iron alum for 10 minutes. Rinse in distilled water and stain in 0.5% aqueous hematoxylin for 1 minute or until darkly stained. Rinse in distilled water and destain in 2% iron alum until a gray color appears. Rinse thoroly in distilled water and intensify hematoxylin by transferring sections to 0.5% aqueous lithium carbonate until the desired black color appears. Rinse thoroly in distilled water and stain for 1-5 minutes in phenolic Bismarck brown. Rinse in distilled water, dehydrate successively in 30, 50, 70, 95 and 100% alcohol. Clear in methyl salicylate for 5 minutes, then to xylene for 3-5 minutes, and mount in balsam.

Middle Lamellae in Wood: Destain more thoroly in 2% iron alum than for the general stem and root schedule, and intensify in lithium carbonate for a longer period (about 1 hour).

White Potato Tuber Sections: Modify above schedule by reducing time of destaining in 2% iron alum to about 30-60 seconds and intensify hematoxylin until starch grains appear bluish in color. Stain in phenolic Bismarck brown for 1-2 minutes.

Wheat Grain Sections: Fix grain for sectioning when in “dough” stage. Use schedule the same as for potato tuber except for reducing time of staining in phenolic Bismarck brown to about 45 seconds.

Tradescantia zebrina Epidermis: Strip epidermis from leaf while submerged in water. Fix in 100% alcohol 10 minutes, pass thru 95, 70, 50, 30, and 10% alcohol to water. Stain in phenolic Bismarck brown for 10-20 minutes. Dehydrate, clear in methyl salicylate and mount in balsam.     

Pyronin Y For Staining Stripped Autoradiographs Prepared from Plastic Embedded Sections of Rat Tissues Containing Plutonium

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.     

Staining Problems with Eosin Y: A Note from the Biological Stain Commission

In 1980, eosin Y was the certified dye with which technologists encountered most problems. The specific problem most frequently brought to the attention of the Biological Stain Commission was that solutions of eosin Y formed a precipitate and failed to stain cytoplasm red when used as a counterstain to hematoxylin.