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.     

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.     

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Abstract

The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.     

A Combined Connective Tissue Stain for Elastin, Reticulin and Collagen

A staining technique for demonstrating reticulin, elastin and collagen in the same tissue sections is based upon the use of a silver stain for reticulin, orcein for elastin and picro-anilin blue or fast green for collagen and other tissue structures.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

Hematoxylin Substitutes: A Survey of Mordant Dyes Tested and Consideration of the Relation of Their Structure to Performance as Nuclear Stains

In the search for hematoxylin substitutes 26 dyes were more or less extensively tested for performance as nuclear stains, usually in combination with aluminum, chromic, ferrous and ferric salts. Reports from the literature on hematoxylin substitutes were also considered, and efforts were made to obtain samples of favorably reported dyes and test them. The reports on anthocyanins include isolated reports on several berry juices and a considerable number of studies on Sambucus niger and Vaccinium mytillus. None of these have so far been tested by us. Otherwise favorable reports have appeared on eleven synthetic dyes and on carmine, brazilin, and hematein. Except for one of the synthetics, naphthazarin, which is no longer manufactured, we had samples of all of these. In addition, more or less unsuccessful trials were made on twelve dyestuffs, some of which were new syntheses designed to combine chelating capacity with nucleophilia. Following Fyg's report of blue nuclear staining with chrome alum carmine, trial was made to change the red nuclear stain of kernechtrot by altering the metal mordant.

The most successful dyes were phenocyanin TC, gallein, fluorone black, alizarin cyanin BB and alizarin blue S. Celestin blue B with an iron mordant is quite successful if properly handled to prevent gelling of solutions.     

Hematoxylin substitutes: a survey of mordant dyes tested and consideration of the relation of their structure to performance as nuclear stains.

In the search for hematoxylin substitutes 26 dyes were more or less extensively tested for performance as nuclear stains, usually in combination with aluminum, chronic, ferrous and ferric salts. Reports from the literature on hematoxylin substitutes were also considered, and efforts were made to obtain samples of favorably reported dyes and test them. The reports on anthocyanins include isolated reports on several berry juices and a considerable number of studies on Sambucus niger and Vaccinium myrtillus. None of these have so far been tested by us. Otherwise favorable reports have appeared on eleven synthetic dyes and on carmine, brazilin, and hematin. Except for one of the synthetics, naphthazarin, which is no longer fractured, we had samples of all of these. In addition, more or less unsuccessful trials were made on twelve dyestuffs, some of which were new syntheses designed to combine chelating capacity with nucleophilia. Following Fyg's report of blue nuclear staining with chrome alum carmine, trial was made to change the red nuclear stain of kernechtrot by altering the metal mordant. The most successful dyes were phenocyanin TC, gallein, fluorone black, alizarin cyanin BB and alizarin blue S. Celestin blue B with an iron mordant is quite successful if properly handled to prevent gelling of solutions.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This adoring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast ells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent wed and the amount of Al²⁺ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Specific Staining of Glycogen with Haematoxylin and Certain Anthraquinone Dyes

Specific staining of glycogen in rat liver fixed in chilled 80% alcohol, chilled formol alcohol or 10% neutral formalin has been accomplished with acid alizarin blue SWR, alizarin brilliant blue BS, alizarin red S, gallein, haematein, and haematoxylin solutions. TO prepare a staining solution, 1 gm dye, 1 gm K₂CO₃ and 5 gm KCl were dissolved by heating in 60 ml of water. Concentrated NH₄OH (0.880 sp.gr.), 15 ml, followed by 15 ml of dry methanol were added to 20 ml of the cooled solution. Paraffi sections were stained for 5 min, rinsed in dry methanol, cleared in xylene, and mounted in D.P.X. The high specificity obviated the need for counterstaining: nuclei and cytoplasm were unstained. Precipitation of stain onto the slide was rare. As all the dyes carried, like carminic acid, numerous groups capable of forming hydrogen bonds, it is suggested that the staining mechanism involved hydrogen bonding.     

Difficulties in Ferrous Iron Mordant Hematoxylin Staining and Some Hematoxylin Substitutes

A gradual deterioration of intensity of sequence ferrous sulfate hematoxylin staining was traced, after elimination of hematoxylin quality as a cause, to a deterioration of the metal salt, associated with caking of the crystals. Fresh samples were also partly caked and ineffective. Ferrous ammonium sulfate was found also subject to the same deterioration. Ferrous chloride freshly prepared as a 1 M solution from iron wire under anaerobic conditions at biweekly intervals proved to be satisfactory as a mordant source. Of several other mordant dyes tested: gallein, brazilin and chromoxane pure blue B were the best, but none was equal to good hematoxylin.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

Staining Myelin in Brain with Gallein : A New Method

A procedure is described in which gallein, mordant violet 25, C.I. 45445, is used to demonstrate myelinated nerve fibers in animal brain. Specimens are fixed in 10% neutral buffered formalin and processed in a routine manner. Microsections are stained in an iron gallein solution with subsequent differentiation in 0.25% oxalic acid and 0.1% sodium carbonate solutions that avoid overdifferentiation. Methyl green is used to demonstrate other tissue elements. Myelin is stained deep violet, as are erythrocytes, with neuronal cell bodies and microglia shades of green. the staining procedure requires 30 minutes.     

Phenylfluorone, a new staining agent with higher selectivity for tin.

In order to search for a new staining agent with higher selectivity for particular metals, the ability of fluorone derivatives to histochemically stain metals present in rat tissues was examined. Among a variety of metals tested, phenylfluorone showed intense staining only for tin. The phenylfluorone method was superior to the conventional gallein method with regard to selectivity when staining for tin among various metals in histochemical practice.     

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G相似文献   

Staining myelin in brain with gallein: a new method.

A procedure is described in which gallein, mordant violet 25, C.I. 45445, is used to demonstrate myelinated nerve fibers in animal brain. Specimens are fixed in 10% neutral buffered formalin and processed in a routine manner. Microsections are stained in an iron gallein solution with subsequent differentiation in 0.25% oxalic acid and 0.1% sodium carbonate solutions that avoid overdifferentiation. Methyl green is used to demonstrate other tissue elements. Myelin is stained deep violet, as are erythrocytes, with neuronal cell bodies and microglia shades of green. The staining procedure requires 30 minutes.