Tests for Nucleic Acid Selectivity of Einarson's Gallocyanin-Chrome Alum Stain on Monolayers of Strain L-929 Mouse Fibroblasts

L-929 fibroblasts, fixed on coverslips, were stained with gallocyanin-chrome alum after various treatments for removal of nucleic acid or for methylation or deamination. For nucleic acid, trichloroacetic acid and NaCl extractions and sequential incubation in DNase and RNase yielded cells unstainable with the dye complex. Methylated cells showed no cytoplasmic staining and a reduced nuclear staining, compared with the unblocked controls. Deamination had little effect. All results were dependent on the types of fixative used, times and temperatures of incubation, and in the case of nucleases, their concentration. Conventional dehydration and melted paraffin infiltration was associated with little or no staining of deaminated cells and intense staining of methylated cells. The paraffin effects were also dependent on fixatives. The evidence shows that gallocyanin-chrome alum combines with groups (presumably phosphate or carboxyl, or both) which are blocked by methylation, and which can be removed from L cells by sequential RNase and DNase treatment.     

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).     

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.     

Nucleoprotein staining. An analysis of a standardized trichloroacetic acid-Fast Green FCF procedure

Synopsis A standardized, highly reproducible modification of the original Alfert & Geschwind procedure (1953) has been formulated; it involves trichloroacetic acid extraction at 60°C and the use of a weak HCl-borate buffer for the dye solution.A comparison of sections stained with Fast Green, Feulgen-Schiff, Toluidine Blue and Gallocyanin-chrome alum indicates that the trichloroacetic acid-Fast Green procedure does not reveal the actual amount of histone but instead simply indicates the excess of positive charges in the chromatin and cytoplasm remaining after the acid extraction.     

蓝莓多糖BBP3-1的分离及结构分析

采用酶法优化进行水提醇沉得到蓝莓粗多糖.经双氧水脱色、酶解和三氯乙酸正丁醇结合法脱蛋白后通过DEAE Sepharose Fast Flow离子交换层析和sephacryl s- 300 HR凝胶过滤层析得到蓝莓多糖(BBP3-1).经紫外扫描显示无蛋白、核酸杂质.经高效液相色谱分析,BBP3-1的重均分子量Mw为18...     

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.     

两种核酸染料染色效果的比较研究

用前染和后染两种不同的染色方法,研究比较SYBRGreenI和溴化乙锭(EB)两种核酸染料对凝胶中DNA的染色效果和灵敏度,及SYBRGreenI取代EB用于常规凝胶中核酸染色的可能性。结果表明,用前染法染色SYBRGreenI对琼脂糖凝胶中的核酸染色效果与EB相当;用后染法染色前者要优于后者,可显示5ng以下的DNA条带,在完全相同的操作条件下,其染色DNA条带背景清晰,灵敏度较高。因此,无致突变性新型染料SYBRGreenI可替代强致突变性染料EB用于检测凝胶中DNA片段大小、含量等,从而减少由于使用EB带来的环境污染和人体健康危害。     

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 demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

Selective Staining of Endocrine Cells by Basic Dyes After Acid Hydrolysis

Staining of tissue sections by basic dyes after immersion in hot hydrochloric acid (0.2 N for 3-10 hr at 60 C) provides a means for selective detection of many endocrine cells. The acid hydrolysis suppresses diffuse basophilia, mainly due to RNA, DNA and acid polysaccharides, and increases the basophilia of secretory granules in endocrine cells, due, at least in part, to the proteins they store. After such treatment, toluidine blue or azur A (0.01-0.005% in 0.02 M McIlvaine buffer, pH 5) or pseudoisocyanin (0.02% in distilled water) heavily stain A and D cells of pancreatic islets, enterochromaffin and nonenterochromaffin endocrine cells of the gastrointestinal mucosa, thyroid parafollicular or C cells, pituitary basophil cells and adrenalin-secreting cells of the adrenal medulla.     

Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Fixation with Bouin's fluid preserves cytoplasmic and nucleolar ribonucleic acid (UNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIIvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin's fluid. This hydrolysis by picric acid permits Schiff staining of the DNA wthout further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.     

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.     

Methods in ultrastructural cytochemistry of the cell nucleus

The electron microscopical study of the cell nucleus as observed in thin sections requires the use of cytochemical methods because of the intricate pattern of the nuclear components. The in situ techniques based on electron staining and enzymatic digestion are reviewed, excluding autoradiography, cytoenzymology and immunocytochemistry. A tentative classification has been adopted according to the chemical nature of the revealed component. Thus, the staining procedures for the nucleoproteins in general, for both nucleic acids, for the proteins, and finally for the deoxyribonucleoproteins and DNA are considered separately. 1--Stains for the nucleoproteins include simple reagents such as the uranyl and lead salts which are largely used in electron microscopy but are of limited specificity. 2--A variety of methods, some of them specific, is available for the simultaneous visualization of DNA and RNA which is based on common properties: basophilia, ability to bind diaminoacridines, presence of hydroxyl groups. However, due to the recent development of specific and preferential methods for each nucleic acid, we feel that among the older methods, only rapid and simple procedures for the detection of both nucleic acids remain of interest. 3--Proteins being ubiquitous, the useful techniques must reveal subsets within the total nuclear proteins. Apart from some endogeneous enzymes, basic proteins -- practically histones -- so far represent the only group for the detection of which reliable methods exist. 4--Several techniques developed recently are available for the specific detection of DNA. In favourable cases, methods derived from the Feulgen reaction allow its visualization at a molecular level. In addition, standard procedures for the preparation of mammalian cells and tissues are described. Each staining method is at least briefly discussed, but emphasis has been placed on a small number of techniques described in detail. They comprise the EDTA regressive stain for the ribonucleoproteins, several reactions of the basic proteins and the Feulgen-like osmium ammine reaction for DNA.     

Polysaccharides from Peptostreptococcus anaerobius and structure of the species-specific antigen

The cell-envelope antigens of Peptostreptococcus anaerobius were extracted from intact cells by autoclave or alkaline treatment. The purified species-specific antigen (G) was identified among several polysaccharides obtained from the extracts by successive treatments with ribonuclease and pronase followed by ion-exchange and gel-filtration chromatography. G was investigated by 13C- and 31P-n.m.r. spectroscopy, titrimetry, elemental analysis, and gas-liquid chromatography. Oxidation of G with NaIO4 followed by reduction with NaBH4 and mild acid hydrolysis yielded the Smith degradation product of G (GS). Treatment of G and GS with 48% HF gave the respective dephosphorylated products GF and GSF. The structures of GS, GF, and GSF were investigated by 13C-n.m.r. spectroscopy, methylation analysis, and gas-liquid chromatography-mass spectrometry. The principal constituents of G were 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), D-glyceric acid, and phosphate as a diester, in the ratio 2:1:1, and a minor amount of D-glucose (beta-D-Glcp). GS contained D-GlcNAc, D-glyceric acid, glycerol, and phosphate in a 1:1:1:1 ratio. GF and GSF contained D-GlcNAc and D-glyceric acid in the ratios 2:1 and 1:1, respectively. A structure for the principal repeating unit of polymeric G compatible with the analytical data consists of alpha-D-GlcpNAc-(1----3)-alpha-D-GlcpNAc-(1----2)-D-glyceric acid units linked through C-6'-C-6" phosphate diester bridges. This structure is novel for two reasons: (a) unsubstituted glyceric acid residues occur as aglycons in the repeating structure, and (b) phosphate diester bridges link nonanomeric glycose carbons in a non-nucleic acid polymer. The structural role of the minor amount of beta-D-Glcp in G remains unknown.     

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.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

Effect of trichloroacetic acid treatment on certain properties of spores of Bacillus cereus T.

Spores of Bacillus cereus T treated with trichloroacetic acid (6.1--61.2 mM) were compared with untreated spores, and as the concentration of the chemical increased, the following alterations in spore properties were found: (1) the extent of germination decreased irrespective of the germination medium used; (2) the spores became sensitive to sodium hydroxide (1 N) and hydrochloric acid (0.27 N), but not to lysozyme (200 micrograms/ml); (3) loss of dipicolinate increased on subsequent heating; and (4) the spores became more sensitive to heat. However, trichloroacetic acid-treated spores were still viable and there was no significant change in spore components. The mechanism of action of trichloroacetic acid is discussed.     

Alkali burns of the rabbit cornea. II. A histochemical study of glycosaminoglycans.

In alkali burned rabbit cornea the stainability of glycosaminoglycans in cold microtome setions was investigated. Staining by Alcian blue in 3% acetic acid, Alcian blue in various MgCl2 concentration and toluidine blue (pH 4.5) was employed. From the 1st to the 4th experimental day the intensity of reactions was decreased. This is most probably due to an increased hydration of the corneal stroma. On the 7th day hydration was markedly suppressed and reached nearly the normal level. In this time interval a decreased stainability of glycosaminoglycans was seen accompanied by a complete loss of staining in the marginal zone. On the 14th day the stainability in the traumatized area began to restore and in the marginal zone appeared. On the 32nd day the staining intensity of both areas was normalised, however when lower concentrations of MgCl2 were used; in the presence of higher concentrations of MgCl2 the decreased staining intensity persisted and points to a lower sulfatation of glycosaminoglycans. This was particularly remarkable in the area bordering the injured zone. This decrease runs parallel to the increased activities of acid glycosidases (especially of acid beta-galactosidase) which were reported previously.