A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.     

Bichromatophilia of rat liver proteins during azo dye carcinogenesis

Summary In the livers of rats fed the azo dye 4-dimethylamino-azobenzene, nucleic acids in connective tissue trabeculae, preneoplastic foci and hepatomas were found to stain intensely with toluidine blue. With the indicator dye bromphenol blue, proteins were observed to stain similarly in these hyperbasophilic tissues but differently from those in surrounding parenchyma.These observations indicate that preneoplastic regions and tumors do not differ from surrounding tissue only by the increased basophilia due to cytoplasmic ribonucleic acid, but also in protein staining. Thus, the change in RNA responsible for hyperbasophila is paralleled by alterations in protein histochemistry. It is suspected that the differential staining of proteins might correspond either to variations in the acidic-basic protein ratios or to the presence of unusual proteins synthesized by such an altered RNA.The tissue similarities in nucleic acid as well as in protein staining observed in the proliferating connective tissue elements and cells undergoing the neoplastic transformation remain an obscure phenomenon.This work was supported by a grant from The Quebec Medical Research Council.     

CYTOLOGICAL AND CYTOCHEMICAL STUDIES OF GREEN MONKEY KIDNEY CELLS INFECTED IN VITRO WITH SIMIAN VIRUS 40

Cytological and cytochemical studies of green monkey kidney cells infected with SV40 virus indicated that the type of lesion produced was influenced by the multiplicity of infection and that the lesions appeared later and progressed more slowly when the inoculum was diluted. The earliest change consisted of enlargement of ribonucleoprotein-containing spherules in the nucleolus (nucleolini). This was followed by rarefaction, with or without condensation, of the chromatin and the appearance of one or more homogeneous masses of inclusion material containing DNA, RNA, and non-histone protein which eventually filled the nucleus. In some instances the chromatin appeared to be directly transformed into inclusion material. In the later stages of infection, the ribonucleoprotein of the nucleolini was no longer stainable and material resembling the nucleoprotein of the intranuclear inclusions was found in the nucleolar vacuoles and in the cytoplasm. The nucleic acids in the inclusions were stained by toluidine blue, toluidine blue-molybdate, the Feulgen stain, and by methyl green. The stainable material was extractable by nuclease digestion or by hot trichloroacetic acid. Green or yellowish green staining by acridine orange was apparently due to binding of dye by protein and not by nucleic acids since the staining reaction was not reduced by extraction of nucleic acids by hot trichloroacetic acid. Extraction with pepsin in combination with ribonuclease or deoxyribonuclease removed practically all the inclusions from the cells; consequently they could not be stained with acridine orange. The cytochemical studies suggest that the use of pepsin together with nuclease is not a meaningful technique.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

The Assessing of Acidophilia with Biebrich Scarlet, Ponceau De Xylidine and Woodstain Scarlet

Horobin and Bennion stated in 1973 that the bonding of Biebrich scarlet at an alkaline pH is hydrophobic as the isomers of Biebrich scarlet (ponceau de xylidine and woodstain scarlet) do not stain at that pH. If correct this would negate the use of Biebrich scarlet as proposed by Spicer and Lillie in 1961 as a measure of acidophilia. We have found all three isomers to stain similarly at an alkaline pH. Goldstein stated in 1963 that Biebrich scarlet saturated with urea stained elastic fibers only faintly or not at all. This we confirmed; we also found that the staining of other acidophilic structures (Paneth cell granules, sperm heads, etc.) was blacked. If only hydrogen bonding and not ionic bonding u blocked in an aqueous dye solution saturated with urea then the exact meaning of alkaline Biebrich scarlet staining is unknown.     

Standardized thionin-eosin stain in bronchial cytology. A substitute for hematoxylin-eosin Y staining

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.     

The assessing of acidophilia with Biebrich scarlet, ponceau de xylidine and woodstain scarlet.

Horobin and Bennion stated in 1973 that the bonding of Biebrich scarlet at an alkaline pH is hydrophobic as the isomers of Biebrich scarlet (ponceau de xylidine and woodstain scarlet) do not stain at that pH. If correct this would negate the use of Biebrich scarlet as proposed by Spicer and Lillie in 1961 as a measure of acidophilia. We have found all three isomers to stain similarly at an alkaline pH. Goldstein stated in 1963 that Biebrich scarlet saturated with urea stained elastic fibers only faintly or not at all. This we confirmed; we also found that the staining of other acidophilic structures (Paneth cell granules, sperm heads, etc.) was blocked. If only hydrogen bonding and not ionic bonding is blocked in an aqueous dye solution saturated with urea then the exact meaning of alkaline Biebrich scarlet staining is unknown.     

A methanol resorcin-fuchsin stain for elastic tissues and nuclei.

The staining properties of conventional ethanol resorcin-fuchsin and of methanol resorcin-fuchsin were compared. Formula; Dissolve 0.2 g of commercial resorcin-fuschin in 70 ml of methanol or ethanol, add 30 ml of water and 1 m1 of concentrated HC1; stain sections for 4 hours. Both solutions colored elastic and pseudoelastic fibers, cartilage and some mucins. Methanol resorcin-fuchsin also colored nuclei in methacarn- (methanol-chloroform-glacial acetic acid 6:3:1) and formalin-fixed tissues; this nuclear stain withstood counterstaining with picro-dye mictures. Zenker-fixed sections showed diffuse coloration with little or no contrast between nuclei and cytoplasm. Extraction with hot trichloracetic acid abolished binding of methylene blue, but binding of methanol resorcin-fuchsin by nuclei remained unaltered or was enhanced. Experiments with solvents containing various concentrations of methanol, ethanol or isopropanol indicated that the staining patterns of resorcin-fuchsin are determined by the nature and concentration of the alcohol. Methanol resorcin-fuchsin proved useful for simultaneous visualization of elastic tissues and nuclei.     

Romanowsky-type staining: Enzymatic analysis of nuclear metachromasia in formalin-fixed paraffin sections

The red color of nuclei produced in formol-fixed paraffin sections stained with toluidine blue has been investigated by using deoxyribonuclease (DNase), ribonuclease (RNase) and 0.1 M Tris buffer. The action of DNase on formol-fixed material is not fully reliable, but clear-cut when positive. Nuclear basophilia and metachromasia is removed, nucleolar and cytoplasmic RNA is preserved. The picture produced by RNase depends to some extent on the concentration and acidity of the toluidine blue used for subsequent staining. Cytoplasmic RNA is always removed, while the red stain in nuclei usually remains intact. With 0.1% toluidine blue in 1% acetic acid, a nuclear color change from red to pale green is observed. Using this same staining solution, it can be shown that 0.1 M Tris buffer (overnight extraction at 37° C) will remove cytoplasmic RNA but leave intact the nuclear material that stains red. A red to green shift can subsequently be produced by RNase. From this it is deduced that there is a chromatin-associated nuclear RNA fraction which can be removed by the enzyme, but is stable to the buffer solution.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

Discriminative Staining Methods for the Nervous System: Luxol Fast Blue-Periodic Acid-Schiff- Hematoxylin Triple Stain and Subsidiary Staining Methods

This paper describes a new series of staining methods which can discrimina-tively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.     

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

Staining Streptomyces Scabies in Lesions of Common Scab of Potato

Toluidine blue can be used to stain Streptotnyces scabies distinctively in slide cultures or in the lesions of common potato scab. This staining method is based on the metachromaticism of volutin, a constant constituent of the spores and mycelium of S. scabies. Either sections or smears are fixed in FPA (formalin, 5 parts; propionic acid, 7.5; 50% alcohol, 87.5), stained in a 1:100 dilution of saturated aqueous toluidine blue from 20 minutes to 24 hours, dehydrated in an acetone-xylene series and mounted. Cellular constituents of the potato tuber stain blue or are colorless whereas the mycelium of Streptomyces appears as a series of red volutin spheres in the blue stained cytoplasm. The criteria of volutin and cytoplasmic staining along with the 1 µ diameter of the mycelium make it possible to distinguish Streptomyces from the other microorganisms and cells in the lesion region.     

HISTONES AND ALKALINE FAST GREEN STAINING OF ONION ROOTS

Onion root tips were freeze killed or fixed in 12 different chemical fixatives to determine the possible effects of such treatments on subsequent staining with alkaline Fast green (pH 8.0-8.1) to reveal the presence of histones. Ethanol, methanol, neutral formalin, and a mixture of methanolchloroform-acetic acid produced nearly comparable results. Subsequent to fixation or freeze drying the sections were treated with hot 5% trichloroacetic acid (TCA) to remove nucleic acids prior to Fast green staining. Staining of histones does not occur in chemically fixed material if TCA hydrolysis is omitted. Nuclei, chromosomes during mitosis, and cytoplasm (to varying degrees) were stainable after TCA treatment. Positive staining occurs in nuclei, nucleoli, and cytoplasm in freeze-dried material with or without prior TCA treatment. Comparisons are made with chemical fixations. The use of ribonuclease instead of TCA permits staining of the cytoplasm and of chromosomes during mitosis. Discussions are included on the stainability of nucleoli and the possibility that native histone is associated with ribonucleic acid (RNA).     

Modification of the method for staining DNA with basic dyes]

The author studied the mechanisms and the applicability in histochemistry of the sodium bisulfate treatment with subsequent toluidine and methylene blue staining after Felgen's hydrolysis. Bisulfite treatment proved to increase the reaction intensity 11/2-fold; the stain is bound stoichiometrically. Toludidine blue results in a metachromatic and anisotropic staining of the cell nuclei. The method is recommended as a sensitive test for DNA in cytochemical investigations and for the study of dichroism of the DNA-containing structures.     

Ethidium bromide: a nucleic acid stain for tissue section

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.