Methyl green-pyronin; basis of selective staining of nucleic acids

1. Methyl green stains selectively highly polymerized desoxyribonucleic acid, and fails to stain, to any significant extent, depolymerized desoxyribonucleic acid and ribonucleic acid. 2. Pyronin stains preferentially low polymers of nucleic acid. 3. Triphenylmethane dyes with two amino groups appear to share the selectivity of methyl green. Those with three amino groups are not selective. 4. A stereochemical hypothesis is offered to account for these observations.     

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 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.     

Methyl green-pyronin; stoichiometry of reaction with nucleic acids

Study of the stoichiometry of the DNA-methyl green reaction by dialysis, precipitation of stain-nucleic acid mixtures, and the staining of nuclei of known DNA content, indicate that the compound consists of one dye molecule per 10 P. The significance of this result was discussed in the preceding paper (1). Histone and lanthanum (and probably other multivalent cations (3)) compete with the dye for the nucleic acid molecule, indicating a common site of attachment, presumably the phosphoric acid groups. With care in the avoidance of procedures which might depolymerize DNA, and the use of a buffer at about pH 4.1, a quantitative histochemical method for DNA by the use of methyl green is possible. Pyronin staining appears to be of qualitative significance only. Slight differences in degree of polymerization, as between the shad and mammalian DNA appear to have no effect on methyl green staining. It may be that a critical level of polymerization for DNA staining exists. This level must exceed 20 nucleotides to account for the 10 P to 1 dye molecule and the effect on the methyl green absorption spectrum; but it may be considerably greater. Beyond this critical level, whatever it may be, further polymerization probably has no influence on staining.     

Methyl green-pyronin stain distinguishes proliferating from differentiated nonproliferating cell nuclei after acid denaturation of DNA

We applied methyl green-pyronin (MG-P) stain, which is usually used for the selective staining of DNA and RNA, to frozen sections of rat jejunal and esophageal mucosa, following digestion with RNase and treatment with various concentrations of HCl. The pyroninophilia of the nuclei increased with increasing strength of the acid, but the susceptibility of the nuclei to acid differed among cell populations. In the jejunal epithelium, at an appropriate acid strength the nuclei in the crypts of Lieberkuhn were less acid-sensitive and remained blue-green, whereas those in the villi were more pyroninophilic and stained lavender. Under the same conditions, the nuclei in the basal layer of the esophageal epithelium were blue-green and those in the spinous and granular layers were increasingly lavender. These results suggest that in cell-renewal systems the differentiated, nonproliferating cells are more sensitive to acid denaturation of DNA than the undifferentiated, actively proliferating cells. MG-P stain, which is able to distinguish double-stranded from single-stranded DNA, may be used as a tool to stain proliferating and nonproliferating cell nuclei differentially in tissue sections.     

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.     

Radiochemical technique for intensification of underexposed autoradiographs

A radiochemical technique has been used to recover images of underexposed and developed autoradiographs. The underexposed image was radioactivated in a solution of [35S]thiourea, air-dried, and reexposed to Kodak NMC film which was developed and processed in a Kodak X-Omat processor. Features which were not discernible in the underexposed autoradiographs were well distinguished in the intensified autoradiograph.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.