Loss of Rna and Protein, And Changes in Dna During A 30-Hour Cold Perchloric Acid Extraction of Cultured Cells

KB cells derived from human carcinoma were fixed in acetic-alcohol (1:3) and extracted with 10% perchloric acid (PCA) at 4 C for 1, 3, 6, 9, 12, 24 and 30 hr. Cells were then washed in water and stained for nucleic acids, proteins, polysaccharides, and lipids. Control cells were kept in water for 30 hr prior to staining. Acridine orange (AO) fluorochroming revealed color changes in residual cytoplasmic and nucleolar RNA as well as DNA during extraction--interpreted as indicative of molecular alterations. All nucleic acid stains (AO, gallocyanin chromalum, and azure B bromide) demonstrated a differential extraction of RNA, with cytoplasmic RNA being removed in about 6 hr and nucleolar RNA requiring 6 more hours for complete extraction. Large granules appeared early in nuclei. These were positive for DNA by azure B, gallocyanin chromalum, Feulgen, and fluorescent-Feulgen. These same granules stained for protein by mercuric bromphenol blue and alkaline Biebrich scarlet. At 24 hr, there was visual and Feulgen-cytophotometric evidence for a slight loss of DNA, which may amount to 10-20%. There was a progressive loss of cytoplasmic and nuclear but not nucleolar protein during PCA treatment. Concurrently, large protein-positive granules appeared in the cytoplasm. Apparently, PCA treatment in combination with an aqueous wash was responsible for some protein loss. Glycogen was gradually lost (fluorescent PAS) and redistributed in cells. Lipids were unaffected (Sudan black B).     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

A selective stain for mitotic figures, particularly in the developing brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

Visualization of viral DNA assembly using nonfluorescent staining

ABSTRACT

We present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.     

Cytophotometric determination of the binding of basic dyes by DNA following acetylation

Summary DNA was removed from various tissues by histochemical acetylation of amino groups in proteins using pure acetic anhydride, as demonstrated by cytophotometric (UV, Feulgen, gallocyanin chromalum) and biochemical techniques. Since new phosphate groups were simultaneously exposed, the intensity of methylene blue staining was increased in spite of the nucleic acid release. Under conditions where no extraction occurs the staining intensity increases for more than 30 per cent. On the other hand, the staining intensity of gallocyanin chromalum kept constant. As it had been demonstrated previously, that gallocyanin chromalum binds to about 86 per cent of the DNA phosphate groups, it was concluded that this dye binds to a higher percentage of phosphate groups than do the usual basic dyes. Since it is not possible under the conditions used to make all nucleic acid phosphate groups available for basic dye binding by blocking the amino groups of proteins it can be assumed that not only electrostatic, but also spatial and steric relationships influence the binding capacity of basic dyes to the phosphate groups of nucleoproteins.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.     

An Evaluation of Cresyl Echt Violet Acetate as a Nissl Stain

The new cresyl echt violet acetate, of which three different batches have been tested, proves to be a very useful Nissl stain. It is especially valuable for formalin-fixed, frozen-sectioned material. By using a buffered staining bath and controlled timing in dehydration it is possible, on paraffin embedded material, to use these dyes as progressive stains apparently specific for nucleic acids. With a saturated aqueous solution of the dye, especially when a mordant of lithium carbonate is used, it is possible to stain material that has been preserved in formalin for several years and also material from which nucleic acids have been removed. The dye is useful also for staining celloidin embedded material. With the buffered stain proposed, differentiation is much easier than with older methods which included a gross overstaining and a long destaining procedure.     

Bismuth Hematoxylin Stain for Arginine Residues

Bismuth ions complex with hematoxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Extraction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of arginine completely abolishes staining.     

Gallocyanin-Chrome Alum: I. Technique and Specificity

Certain technical aspects of gallocyanin-chrome alum were examined relative to its supposed specificity for nucleic acids. Five different lake formulae were prepared using four different batches of gallocyanin. Spectrophotometric curves were made of each lake and of each dye in a simple water solution. Paraffin sections 6-8 μ thick of spinal cords from albino rats and from cats fixed in CaCl₂-formalin or plain formalin were stained 10 min to 48 hr with gallocyanin lakes made with chrome alum, ferric alum, strontium chloride and copper nitrate. Similar sections were treated with ribonuclease or perochloric acid and stained in the same manner. The spectrophotometric data indicates considerable variation in dye content between different batches and different lakes. Chrome alum was the best of the 4 mordants and a 12-15 hr staining time with Einarson's 1932 preparation was optimal. Neither perchloric acid nor ribonuclease destroyed cytoplasmic basophilia as revealed by gallocyaninchrome alum. Staining was more intense after CaCl₂-formalin fixation than after plain formalin. Variation of the dye content in the different batches of dyes, the poorly understood role of boiling in preparing the lakes, and the inability of ribonuclease or perchloric acid to destroy cytoplasmic basophilia indicates that we are not dealing with a histochemically specific reagent for nucleic acid, but only a desirable nuclear stain.     

The staining of mucopolysaccharides with gallocyanin and metal mordants

Summary Some salts of tin, titanium, zirconium, hafnium, niobium, tantalum, molybdenum and tungsten can act as mordants which can combine with polysaccharides and their presence can be detected as colored compounds with an acid alcoholic solution of gallocyanin. None of these metals produced color reaction when used alone. Titanium, zirconium, hafnium, niobium, tantalum and tin form a blue gallocyanin color with salivary, laryngeal, bronchial, gastrointestinal, uterine cervical gland mucins, cartilage and rat mast cells. In addition, tin (IV), titanium (IV), hafnium, niobium and tantalum stained Brunner gland bluish violet to a purple. Gallocyanin mordanted with molybdenum and tungsten stained collagen, reticulum and cartilage blue to dark blue. In addition, tungsten stained some elastic fibers a bright red violet. Paneth cells were stained dark blue by niobium, molybdenum and acidified titanium potassium oxalate. Molybdenum could be extracted by alkalis and titanium by strong acids after tissues have been mordanted. Methylation prior to mordanting inhibits staining of the mucosubstances by titanium. Sulfation enhances staining reactions by titanium and molybdenum.     

A Modified Turchini Technic for the Differential Staining of Nucleic Acids

The preparation of the 9-methyl-2,3,7-trihydroxy-6-fluorone reagent for the selective staining of both desoxyribose and ribose nucleic acids is described. With slight variations this method follows the Duckert (1937) modification of the Liebermann and Lindenbaum (1904) reaction.

The present modification of the Turchini et al. (1944) staining procedure has been used on human autonomic ganglia fixed in Bouin's fluid, rat tissues, fixed in Bouin's, Zenker's, formol and for-mol-saline fixatives and mouse liver frozen and dried. The modified Turchini method has been examined primarily for its qualitative reliability by means of the following procedure. Ribonuclease treated sections were compared with adjacent sections immersed in distilled water. In succeeding steps half of these sections were stained by the modified Turchini process and the other half by Einarsson's gallocyanin chrome alum. Evidence gleaned from this and other tests indicates that 9-methyl-2,3,7-trihydroxy-6-fluorone may be used for the selective staining of desoxyribose and ribose nucleic acids.     

Electron spectroscopic imaging of chromatin.

The analytical electron microscope technique called electron spectroscopic imaging (ESI) has a number of applications in the study of DNA:protein complexes. The method offers an intermediate level of spatial resolution for in vitro structural studies of complexes that may be too large or heterogeneous to study by crystallography or magnetic resonance spectroscopy. An advantage of ESI is that the distribution of nucleic acids can be resolved in a nucleoprotein complex by mapping the element phosphorus, present at high levels in nucleic acid compared to protein. Measurements of phosphorus content together with mass determination allows estimates to be made of stoichiometric relationships of protein and nucleic acids in these complexes. ESI is also suited to in situ studies of nuclear structure. Mass-sensitive images combined with nitrogen and phosphorus maps can be used to distinguish nucleic acid components from nuclear structures that are predominantly protein based. Interactions between chromatin on the periphery of interchromatin granule clusters (IGC) with the protein substructure that connects the exterior of the IGC to its core can be studied with this technique. The method also avoids the use of heavy atom stains, agents required in conventional electron microscopy, that preclude the distinguishing of structures on the basis of their biochemical composition. The principles of ESI and technical aspects of the method are discussed.     

FINE STRUCTURAL AND RADIOAUTOGRAPHIC OBSERVATIONS ON DENSE PERINUCLEAR CYTOPLASMIC MATERIAL IN TADPOLE OOCYTES

Dense fibrous material is first seen in association with mitochondria in tadpole oogonia but is most prominent in oocytes during the extended first meiotic prophase when it aggregates into dense bodies in the perinuclear cytoplasm. The origin of this material has been attributed to 350-A nuclear granules which form cytoplasmic streamers of fibrous material upon passage through nuclear pores. This has commonly been interpreted as the transfer of ribonucleoprotein to the cytoplasm for storage. However, cytochemical reactions for nucleic acids have indicated an absence of detectable RNA in this dense material, and the results of radioautographic studies with labeled uridine, thymidine, or actinomycin D argue against the presence of nucleic acids. When sites of incorporation of tritiated amino acids were radioautographically localized, an appreciable number of silver grains were present over the dense bodies. Uptake of certain amino acids occurs fairly promptly but the degree of labeling levels off after about 6 hr, suggesting a rapid turnover of the material in the dense bodies. Attention is drawn to the similarity of the dense bodies to structures present in germ cells of a number of other species, and possible functions of the dense bodies in germ cell differentiation are considered.     

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 effect of graded 60 degrees C 1N nitric acid extraction and of deoxyribonuclease digestion on nuclear staining by metachrome mordant dye metal salt mixtures.

We can divide metachrome mordant staining of nuclei after graded 60 degrees C 1 N nitric acid extraction into three groups. The Feulgen nucleal reaction and dilute cationic dye staining of nuclei are abolished in about 30 minutes. With one group of metachrome dyes nuclear staining is lost with acid exposures of one hour or less. In a second group nuclear staining is weakened by 30-60 minute extractions, but persists in recognizable grade for 4-6 hours. In the third group nuclear staining remains almost unimpaired for 4-6 hours. In the first group the nuclear staining seems clearly assignable to the nucleic acids and to DNA in particular. In the second group loss of part of the reactivity on short exposure indicates some participation of DNA in the control staining result, as well as participation of basic nucleoprotein. In the third group staining seems assignable largely to basic nucleoprotein. The five gallocyanin group dyes, all in group 1, all possess a dialkylamino group, probably functioning as an ammonium chloride.Hematoxylin, the flurone blacks and gallein all present an o-hydroxysemiquinone group which probably acts as a weak acid, in addition to the carboxyl group of gallein which gives the strongest staining of nuclei at the longest acid exposure. Deoxyribonuclease digestion (2 hours, 37 degrees C) separated sharply a class in which nuclear staining failed completely, a class in which nuclear staining was fully equal to that in the control preparations and an intermediate group in which slight, moderate, or severa impairment was present. Generally there was good agreement between the two methods of nucleic acid removal, despite the fixation difference. In each case, however, the extraction procedure was one worked out for the fixation on which it was used.     

Hematoxylin substitutes: fluorone black and methyl fluorone black (9-phenyl- and 9-methyl-2,3,7-trihydroxy-6-fluorone) as metachrome iron alum mordant dyes.

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini technics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nucleic acids, as with hematoxylin lakes. The two dyes, named by Liebermann and Lindenbaum 9-phenyl-2, 3, 7-trihydroxy-6-fluorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2,6,7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog. The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl flurone black less useful. Neither dye has the diverse capability of hematoxylin.     

White gels: an easy way to preserve methylene blue stained gels

Methylene blue can be used as a stain for visualizing nucleic acids in polyacrylamide gel electrophoresis. However, its relatively low sensitivity and reversible binding make it a temporary stain that diffuses from the gel relatively fast. Here we describe a very simple method for fixing methylene blue bands in nucleic acid polyacrylamide gels. The procedure makes the methylene blue stain permanent and increases the visibility of the bands, also contributing to increasing the sensitivity of methylene blue.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.