Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Chlorous Acid as An Agent for Blocking Tissue Aldehydes

Chlorous acid (HClO₂), which is known to convert aldehyde groups to carboxylic groups, was found to be an effective blocking agent for tissue aldehydes. A 0.2 M solution of NaClO₂ in 1 M acetic acid was found to block completely the Feulgen, PAS, ninhydrin-Schiff and plasmal reaction within 8 hr at 20 C. This blocking reaction appears to be irreversible.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

Rapid method for the determination of lysine in cereal grains without hydrolysis.

A rapid spectrophotometric method for lysine determination in cereal grains is reported. The reagent is sodium dinitrobenzene sulfonate (DNBS). The absorbance of the acid solution of the DNBS derivative is read at 385 nm. Under prescribed conditions, the reagent is sensitive and specific for free or protein-bound lysine. The conditions: For rice, the reaction medium is a urea-phosphate-HgCl₂ solution (pH 10.5); reaction at 60°C for 1 hr. For other grains, the reaction medium is a urea-phosphate-phenylmercuric chloride (PMC) solution (pH 12.3); reaction at 40°C for 1 hr. Interferences are eliminated by ether extraction after color development and acidification and addition of formic acid and the sulfhydryl masking agents, HgCl₂ or phenylmercuric chloride (PMC). NaOH extracts of cereal proteins are used for analysis. Values are in agreement with those of the ion-exchange amino acid analyzer.     

Measurement of DNA content in single cells morphologically identified on smears

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.     

The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999     

Feulgen hydrolysis and chromosome banding in Chilocorus (Coleoptera: Coccinellidae).

Prolonged Feulgen hydrolysis of chromosomes of Chilocorus orbus Csy. and C. stigma Say produces banding patterns that are the reverse of those revealed with quinacrine; brightly fluorescing regions are unstained, but nonfluorescent regions remain relatively darkly stained. This differential reactivity at hydrolysis times that otherwise yield intense Feulgen staining confirms the need for caution in the determination of DNA values with the Feulgen reaction in material with well-defined quinacrine bands. The coincidence of DNA-specific Feulgen bands with Q-, G-, and C-bands supports the view that, in Chilocorus at least, bands reflect differences in DNA composition along the chromosome.     

Critical analysis of the use of the acrolein-Schiff method as a possible DNA reaction

Summary Histophotometric examination was carried out on nuclei of lymphocytes in human peripheral blood, which were subjected to various tests in order to assess the acrolein-Schiff method as a possible DNA specific reaction, in comparison with the traditional Feulgen reaction. Special attention was paid to the degree of difference between responses attributable to a direct Schiff reaction obtained in the fraction of nuclear proteins after treatment with acrolein. From the results obtained it appears that an acrolein-Schiff reaction, following extraction of proteins, may be considered a qualitative reaction for DNA. Our findings also show that there is no relationship between the degree of response to the acrolein-Schiff reaction and that to the Feulgen reaction, which is to be expected in view of the different mechanisms of the two reactions.     

Texture formation in DNA films with alkali metal chlorides

The formation of textures in DNA films with LiCl, NaCl, KCl, RbCl, and CsCl salts has been studied. The films are prepared by evaporation of water solution with highly polymerized calf thymus DNA and excess salt of specific type. For DNA solution with 10 mM concentration of NaCl, KCl, and RbCl the films with dendritic textures have been obtained, whereas in case of CsCl the textures in the films appear only at 30 mM concentration of excess salt in the initial solution. In the solution with LiCl, the textures in DNA films have not been observed within the whole range of concentration of excess salt under consideration. The analysis of parameters of DNA films with different salts has showed that evaporation of solution leads to crystallization of salt ions on DNA macromolecule and formation of DNA‐salt complexes. Electrostatic energy of the system of crystalline ordered ions and charges of DNA chains has been estimated to study the stability of DNA‐salt complexes. The results obtained for different salts have been showed that the presence of DNA macromolecule enhances crystallization as compared with solution without DNA. The property of excess salt to form the crystalline structures has been found to decrease in the following order: KCl > NaCl > RbCl > CsCl > LiCl. The results of estimation are in good agreement with the experimentally observed dependence of texture formation on excess salt type. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 508–516, 2013.     

实验性肝硬化部分肝切除后肝组织学及组织化学变化的研究

四氯化碳所致肝硬化的大白鼠,进行７０％和３０％肝脏切除,于术后４８小时、１周、２周分别取剩余肝脏观察肝的组织化学变化,包括ＤＮＡ（脱氧核糖核酸）,ｈｉｓｔｏｎｅ（组蛋白）、ＲＮＡ（核糖核酸）、ＳＤＨ（琥珀酸脱氢酶）、Ｇ－６－Ｐａｓｅ（葡萄糖－６－磷酸酶）、Ｍｇ－ＡＴＰａｓｅ（镁激活三磷酸腺苷酶）、ＣｈＥ（胆碱酯酶）、ＡＫＰ（碱性磷酸酶）、ＡＣＰ（酸性磷酸酶）,ＰＡＳ（糖原）反应。其中肝硬化切除３０％肝脏的动物,术后４８小时再生肝细胞活跃,肝脏ＤＮＡ、ｈｉｓｔｏｎｅ、ＳＤＨ、Ｇ－６－ｐａｓｅ、ＡＴＰａｓｅ活性及反应明显增高;而ＣｈＥ活性及ＰＡＳ反应等显著减弱。     

Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction

The possibility of using the Feulgen nucleal reaction for a quantitative cytochemical estimation of desoxyribonucleic acid (DNA) was investigated. The intensity of the reaction in nuclei was determined by absorption measurements with the microscope. The accuracy of such measurements was tested by comparison with measurements on the same material with a Beckman spectrophotometer. The values obtained with the microscope agreed within a few per cent with those obtained with the Beckman spectrophotometer. Furthermore, the errors introduced by uneven distribution of absorbing material, by variations in the numerical aperture of the system, and by variation in the area used on the phototube were investigated empirically. The following variables were studied with regard to their effect on the intensity of the Feulgen reaction: type of fixation, time of hydrolysis after acetic acid-alcohol and formalin fixation, time of staining in leucobasic fuchsin, method of preparation of leucobasic fuchsin. The intensity of the Feulgen reaction in liver and erythrocyte nuclei of various vertebrates, fixed in acetic acid-alcohol, was then compared with the DNA content of these nuclei as determined by chemical analysis on a known number of nuclei. The intensity of the reaction was found to be proportional to the DNA content of the nuclei, if nuclei of similar structure and DNA concentration were compared. In nuclei of different structure and DNA concentration (i.e. liver and erythrocyte nuclei), fixed in acetic acid-alcohol, the intensity of the Feulgen reaction was, however, not proportional to the DNA content. This difficulty was overcome by isolating nuclei in sucrose and by fixing them in formalin. Uniform distribution of DNA and therefore uniform coloring after the Feulgen reaction were thus obtained. In such nuclei with uniform distribution of absorbing material the Feulgen reaction was found to be proportional to the DNA content of nuclei, even if they differed greatly in their DNA concentration. The Feulgen nucleal reaction is not quantitative in an absolute sense. For absolute determinations nuclei of known DNA content must be treated together with the unknown material to serve as standard. From these data it therefore appears possible to determine cytochemically relative amounts of DNA in cellular structures by measuring their absorption after treatment with the Feulgen nucleal reaction.     

Quantification of nuclear DNA and intracellular glycogen in a single cell by fluorescent double-staining.

It was found that intracellular glycogen is stabilized against acid treatment when it is stored under dry conditions for three months after methanol fixation. This stabilization allowed quantitative double fluorescence staining for nuclear DNA and intracellular glycogen, in a single cell. A Feulgen nucleal reaction, with acriflavine-Schiff's reagent following 5 N HCl hydrolysis at 25 degrees C for 4 min, was followed by a pararosanilin-Schiff PAS reaction for glycogen. This short term hydrolysis was found to be sufficient for the performance of a acriflavine-Schiff's Feulgen nucleal reaction and to provide good preservation of intracellular glycogen. Quantification of nuclear DNA and intracellular glycogen were consecutively carried out with a digital microfluorometer on a single ascites cancer cell of the AH-13 line stained by this method. It was found that there is a positive linear correlation between the amount of DNA and glycogen in this cell line.     

Feulgen Cytophotometry of Pine Nuclei; Effects of Fixation, Role of Formaline

The effects of 5 fixatives: FAA, Carnoy's, Craf III, formalin and glutaraldehyde were analyzed for use in quantitative Feulgen cytophotometry of pine embryo tissues. Craf III and glutaraldehyde had serious deficiencies because they depressed the absorption peak, severely interfered with DNA extraction and in the case of glutaraldehyde there was considerable cytoplasmic dye-binding. Neutral 10% formalin gave good tissue fixation but did not permit the degree of enzymatic or acid extraction of DNA as did Carnoy's solution. Haupt's adhesive, with the usual 4% formalin as a hardener, at temperatures of 45-56 C completely prevented the enzymatic extraction of nuclear DNA by DNase and also greatly increased the resistance of the DNA to mineral acid hydrolysis. Denaturation of DNA by formalin appeared to be responsible for these results. Absorption was linearly related to both section thickness and DNA concentration per nucleus.     

DNA content in the mouse spermatogonia B and spermatid nuclei during treatment with insecticides

The DNA content in nuclei of germ cells: spermatogonia B and spermatides in the maturation phase of male mice receiving commercial insecticide preparations commonly used in this country was cytophotometrically measured. The following insecticides were tested: 1% Metox-30 solution, containing 30% of methoxychlorine (p,p-dimethoxydiphenyltrichlorethane), 0,3% solution of Sadofos-30 containing 30% of malathione (1,2-dicarboethoxyethyl-0,0-dimethyldithiophosphate) and 0,3% solution of Foschlor-50 containing about 50% trichlorfon (2,2,2-trichloro-1-hydroxyethyl-0,0-dimethyl-diphosphonate). It was found that a statistically significant number of spermatogonium B and spermatide nuclei in the phase of maturation of the animals treated with the insecticides exhibited an abnormal DNA content as compared with the nuclei of control animals. The chromatin of these nuclei is also more sensitive to acid hydrolysis in Feulgen reaction.     

Quantification of nuclear DNA and intracellular glycogen in a single cell by fluorescent double-staining

Summary It was found that intracellular glycogen is stabilized against acid treatment when it is stored under dry conditions for three months after methanol fixation. This stabilization allowed quantitative double fluorescence staining, for nuclear DNA and intracellular glycogen, in a single cell. A Feulgen nucleal reaction with acriflavine-Schiff's reagent following 5 N HCl hydrolysis at 25°C for 4 min, was followed by a pararosanilin-Schiff PAS reaction for glycogen. This short term hydrolysis was found to be sufficient for the performance of a acriflavine-Schiff's Feulgen nucleal reaction and to provide good preservation of intracellular glycogen. Quantification of nuclear DNA and intracellular glycogen was consecutively carried out with a digital microfluorometer on a single ascites cancer cell of the AH-13 line stained by this method. It was found that there is a positive linear correlation between the amount of DNA and glycogen in this cell line.This work was partly supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

Increased cytolytic T lymphocyte activity induced by 2-mercaptoethanol in mixed leukocyte cultures: kinetics and possible mechanisms of action.

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 muM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period. The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulphydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products. Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 nad 72 hr after the onset of MLC. When 2-ME was added to day-3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue"period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.     

The effect of HCl hydrolysis on the retention of thymidine in DNA

Allium roots grown in C(14)-thymidine and H(3)-thymidine media were treated with N hydrochloric acid at 60 degrees C. as in standard Feulgen hydrolysis. The retention of the radioactive thymidine in DNA as a function of hydrolysis time was studied autoradiographically. No significant loss of label was detected until hydrolysis was extended beyond the optimal time for Feulgen staining. The data are consistent with the assumption that there is no significant loss of DNA during normal Feulgen hydrolysis in the material used.     

A fluorescent histochemical method for the detection of tissue disulphide groups

Summary Bennett and Yphantis (1948) introduced into histochemistry the use of organic mercurials for the detection of sulphydryl groups. The new fluorescent method which is now reported is similarly based upon the reaction of mercaptans with an organic mercurial, fluorescein mercuric acetate (FMA). This method is sensitive; moreover it is not as elaborate as the dihydroxy-dinaphthyl-disulphide (DDD) technique of Barrnett and Seligman (1952).