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).     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.     

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.     

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

Summary We can divide metachrome mordant staining of nuclei after graded 60°C 1N 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 group1, all possess a dialkylamino group, probably functioning as an ammonium chloride. Hematoxylin, the fluorone blacks and gallein all present ano-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. Dexyribonuclease digestion (2 hours, 37°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. Assisted by National Cancer Institute Contract No. NO 1 CB43912.     

Groove-binding unsymmetrical cyanine dyes for staining of DNA: syntheses and characterization of the DNA-binding

Two new crescent-shaped unsymmetrical cyanine dyes have been synthesised and their interactions with DNA have been investigated by different spectroscopic methods. These dyes are analogues to a minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a benzothiazole substituent. To investigate how the identity of the extending heterocycle affects the binding to DNA, the new dyes BETO and BOXTO have a benzothiazole group and a benzoxazole moiety, respectively. Whereas BEBO showed a heterogeneous binding to calf thymus DNA, linear and circular dichroism studies of BOXTO indicate a high preference for minor groove binding. BETO also binds in the minor groove to mixed sequence DNA but has a contribution of non-ordered and non-emissive species present. A non-intercalative binding mode of the new dyes, as well as for BEBO, is further supported by electrophoresis unwinding assays. These dyes, having comparable spectral properties as the intercalating cyanine dyes, but bind in the minor groove instead, might be useful complements for staining of DNA. In particular, the benzoxazole substituted dye BOXTO has attractive fluorescence properties with a quantum yield of 0.52 when bound to mixed sequence DNA and a 300-fold increase in fluorescence intensity upon binding.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

The binding of aminoacyl-tRNA synthetases to triazine dye conjugates.

The binding of thirteen aminoacyl-tRNA synthetases to thirty two immobilised procion dyes has been investigated. Most dyes bind one or more enzymes. The amino acid substrates are not normally potent eluants, with the notable exception of tryptophan eluting tryptophanyl-tRNA synthetase from Brown MX-5BR. Phosphate is frequently extremely effective, much more than expected by simple considerations of ionic strength, indicating that many of the dyes are able to mimic the phosphate groups of the phosphodiester backbone of the nucleic acid. Procedures for the purification of methionyl-, tryptophanyl- and tyrosyl-tRNA synthetases are presented and compared to the conventional purifications of these enzymes. The results indicate the general applicability of these dye columns to the purification of most enzymes of of nucleic acid metabolism and the necessity of investigating as many different dyes as possible for any individual enzyme.     

Anisotropic staining of apurinic acid with toluidine blue

Summary Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Anisotropic staining of apurinic acid with toluidine blue.

Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

Some observations on the mechanisms of blocking of nuclear staining by cisplatin

Summary The effect of cisplatin (cis-dichloro-diamminoplatinum II) treatment on staining of nuclei with various basic dyes and with the Feulgen reaction has been studied. Although cisplatin is reported to show negligible reaction with DNA phosphates, it has a substantial blocking effect on staining with most dyes. Short treatment with cisplatin results in binding mainly to guanine bases of DNA, causing partial blocking of the Feulgen reaction and almost complete blocking of ethidium intercalation; binding of neutral red and crystal violet is enhanced, apparently as a result of cisplatin-induced denaturation of DNA. Very prolonged cisplatin treatment does not completely block the Feulgen reaction, indicating that reaction of cisplatin with purine bases is not complete. Since attachment of cisplatin to DNA bases is unlikely to prevent binding of most basic dyes, it is suggested that the blocking of their staining may result from steric hindrance caused by formation of DNA-protein cross-links by cisplatin. Whatever the mechanism, it is incapable of producing complete blocking of staining with certain dyes. As a practical tool, it appears that rapid and almost complete blocking of staining by cisplatin may be used as an indicator of intercalative binding of dyes to DNA.     

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

Summary 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 analtion. 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.     

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.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

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.     

The circular dichroism of complexes of 2,7-di-tertiary-butyl proflavine with DNA

The binding isotherm of 2, 7-di-tert-butyl proflavine on calf thymus DNA has been measured by dialysis equilibrium. The CD spectra of complexes of the dye and DNA have been measured, and the variation of the induced circular dichroism of the dye with the amount of dye bound (r) has been found. The results show that di-tert-butyl proflavine binds to DNA in a completely different manner from proflavine itself, since both the visible and ultraviolet CD spectra of complexes of the two dyes with DNA differ markedly. The conformation of the nucleic acid is not affected by the binding of di-tert-butyl proflavine. It is possible that these results may allow determination, by using CD spectroscopy, of whether molecules intercalate into DNA.     

Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding.

1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.     

Influence of some aldehyde blocking agents on staining of depurinized DNA with cationic dyes.

Rat liver, spleen and Walker carcinosarcoma imprints were subjected to depurinizing Feulgen hydrolysis and then treated with blocking agents of aldehyde groups. Such blockators as sodium bisulfite and hydroxylamine which multiplay additionally anionic groups in DNA and intensify the reactions with cationic dyes, ensuring anisotropic staining. Hydrazine lowers the binding of carionic dyes to DNA, instead phenylhydrazine, completely blocks both aldehyde and phosphate groups. When the imprints were treated with 2.4-dinitrophenylhydrazine, aldehyde and phosphate groups of apurinic acid were blocked, and DNA staining by cationic dyes occurred only on account of nitrogroups of the blocking agents which have been used. The staining reaction of cationic dyes after the use of anionogenic blocking agents of aldehyde groups is prospective not only for revealing DNA but also for several other compounds with natural or potential aldo- and ketogroups. However the reaction with phenylhydrazine can serve as a staining without removal of DNA prior to staining as an optional procedure.