Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.     

The Determination of Apparent Isoelectric Points of Cell Structures by Staining at Controlled Reactions

The staining reactions at controlled pH-values of various dyes with the nucleus and cytoplasm of Trichonympha collaris under different conditions were investigated. When staining intensity was plotted against pH, it was found that with each dye a different curve was obtained. “Isoelectric points” obtained by superposition of acid and basic dye curves varied for the same material with the dyes employed. It was found that, with the same dye, the curves of staining intensity plotted against pH varied with the buffer system utilized. Moreover, the intensity of staining at any pH was found to vary directly with the concentration of dye and inversely with the concentration of buffer. Various factors modifying staining intensity were studied. In the staining of a protein in buffered solution, it was shown that staining intensity (the index of the concentration of the dye-protein compound) at a given pH-value is dependent upon the interaction of the dye-protein, buffer-protein and dye-buffer systems, and that as the dye or buffer or their concentrations were varied, the resultant “isoelectric points” which were obtained also varied. In view of these facts and of the present lack of knowledge of dyes and dye-protein combinations it would be impossible to determine a true isoelectric point by staining at controlled pH-values without further extensive work on the subject. It follows that no true isoelectric points have hitherto been obtained for nucleus, cytoplasm or other tissue elements by staining at controlled pH.     

Staining of keratin and keratohyalin with the reactive dye levafix red violet E-2BL.

Demonstration of keratin in Zenker-fixed skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-2BL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fixed sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaCl, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Staining of Keratin and Keratohyalin with the Reactive Dye Levafix Red Violet E-2BL

Demonstration of keratin in Zenker-fired skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-PBL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fired sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaC1, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Comparisons of Pyronin Dyes Obtained from Various Commercial Sources. 1. History

The history of pyronin dyes is discussed, beginning with the synthesis of pyronin Y(G) in 1889. The chemical structures of the dyes are given in addition to references to literature describing methods of synthesis. The early histological use of pyronins is described as well as the distribution and use of pyronins in the U. S. A. and Europe. Further discussion is devoted to the use of pyronins for histochemical demonstration of ribonucleic acid, especially in relation to sources, dye variability, contamination, and substitution by rhodamines. Additional studies with improved pyronins are advocated to evaluate histochemical staining mechanisms and to investigate possible quantitative uses.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

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.     

Prediction of in situ fluorescence of histochemical reagents using a structure-staining correlation procedure

Summary A total of ninety acid, basic, and non-ionic dyes were screened for fluorescent staining of various Carnoy fixed rat tissues. It was found that the fluorescence/nonfluorescence of a dye could be predicted using a conjugated bond number (CBN) cut-off value. Thus 90% of dyes with CBNs of 29 or less were fluorescent; whilst 70% of dyes whose CBNs exceeded 30 were nonfluorescent. The cut-off value was not significantly influenced by the charge, or the hydrophobic-hydrophilic character of the dye; though fluorescence was greatly influenced by the mode of fixation. The CBN cut-off value proved surprisingly robust. Thus most fluorochromes found in the histochemical literature have small conjugated systems, with CBNs less than the cut-off value. This includes labels of immunoglobulins, vital stains of neurones, and fluorescent Schiff reagents. Conversely several dyes used to quench background autofluorescence have large conjugated systems, with CBNs substantially above the cut-off value.In honour of Prof. P. van Duijn     

Prediction of in situ fluorescence of histochemical reagents using a structure-staining correlation procedure

A total of ninety acid, basic, and non-ionic dyes were screened for fluorescent staining of various Carnoy fixed rat tissues. It was found that the fluorescence/non-fluorescence of a dye could be predicted using a conjugated bond number (CBN) cut-off value. Thus 90% of dyes with CBNs of 29 or less were fluorescent; whilst 70% of dyes whose CBNs exceeded 30 were nonfluorescent. The cut-off value was not significantly influenced by the charge, or the hydrophobic-hydrophilic character of the dye; though fluorescence was greatly influenced by the mode of fixation. The CBN cut-off value proved surprisingly robust. Thus most fluorochromes found in the histochemical literature have small conjugated systems, with CBNs less than the cut-off value. This includes labels of immunoglobulins, vital stains of neurones, and fluorescent Schiff reagents. Conversely several dyes used to quench background autofluorescence have large conjugated systems, with CBNs substantially above the cut-off value.     

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   

ISOELECTRIC POINTS FOR THE MYCELIUM OF FUNGI

1. Mycelium of Rhizopus nigricans when stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.0. 2. When grown on potato dextrose agar the reaction of which was varied with phosphoric acid the extent of colony growth of Rhizopus nigricans plotted against the initial Sörensen value of the agar produced a double maximum curve with the minimum between the two maxima at initial pH 5.2. 3. When grown in potato dextrose broth the reaction of which was varied with phosphoric acid the dry matter produced by Rhizopus nigricans plotted against the Sörensen value of the broth produced a double maximum curve with the minimum between the two maxima at initial pH 5.2 or average pH 4.9. 4. Mycelium of Rhizopus nigricans placed in buffer mixtures of 0.01 M phosphoric acid and sodium hydroxide of pH 4.1 to 6.3, changed the reaction in most cases toward greater alkalinity. 5. Mycelium of Fusarium lycopersici stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.5.     

Direct recording of metachromatic spectra in a model system of polyacrylamide films

Synopsis The metachromatic staining of polyacrylamide films containing different glycosaminoglycans is described. This model system made direct recording of metachromatic curves possible, under circumstances comparable to those of stained sections under the microscope, with virtually no interference of the corresponding orthochromatic peaks.The staining was carried out under standardized conditions (of buffer concentration, pH and temperature) and was shown to follow the Lambert-Beer law. The metachromatic peaks obtained with this system are listed for seven basic dyes, each complexed with seven different glycosaminoglycans.     

Deamination to prevent the competitive effect of proteins in the metachromatic staining of sections at low pH-values

Summary To prevent the competitive cation effect of present proteins in the utilization of the effect of pH in differential histochemical staining of mucopolysaccharides with cationic dyes, preliminary oxidative deamination with 0.5% ninhydrin in absolute ethanol at 37° C was carried out. Thirty minutes deamination was sufficient to prevent the competitive effect of proteins at low pH values (1.0), even in tissue (cornea) with a high content of proteins compared with the content of acid mucopolysaccharides (glycosaminoglycuronoglycans).This work has been supported by grants from the Danish State Research Foundation.     

Electrophoretic and histochemical studies of carbonic anhydrase activity

Summary A simple method for separation of carbonic anhydrase activity into components by electrophoresis on cellulose acetate strips is described. With this method, using barbiturate buffer systems at various pH values, two main components of CAH in rat erythrocytes, and the splitting of each of these into two minor components were revealed. Two components were also observed in the CAH activity in kidney and lens homogenates, and one component in brain homogenate. A modification of Häusler's histochemical method for CAH was adapted for visualization of the electrophoretically separated bands. This rendered the evalution of the results easier than with the quantitative measurements alone. The quantitative measurement of CAH activity in electrophoretic strips corresponded with the degree of staining by the histochemical method. This among other facts supports the view of the specificity of the histochemical method used. Some examples of the histochemical staining pattern of the CAH activity in rat tissues are given.     

Staining of Mucus with Buffered Solutions of Toluidine Blue O, Thionin and New Methylene Blue N

The addition of buffer mixtures to toluidine blue O, thionin and new methylene blue N improves their use in the staining of mucus after formaldehyde fixation. Overstaining is minimi The buffer mixtures used consisted of variable proportions of M/10 citric acid and M/5 anhydrous Na²HPO₄ in 25% methanol. Connective tissue mucus stained satisfactorily with these dyes at a buffer pH range of 3.4 and 3.95 and epithelial mucus at a range of 2.2 to 3.95. The corresponding ratios of M/10 citric acid: M/5 Na₂HPO₄ are 16:4 to 14:6 and 20 A to 14:6 respectively.     

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.     

Controlled Staining of Autoradiographs

The preparation of autoradiographs in which the tissue and the emulsion are in permanent register is often complicated by staining after the photographic image has been developed and fixed. While general oversight methods can be satisfactory, controlled, specific staining can be obtained with most basic dyes when the pH is properly regulated. The reactivity of the gelatin is suppressed at a pH of 4 or slightly below whereas nuclei, ergastoplasm, cartilage, mast cells, mucus, etc. stain readily. Basic fuchsin, .05% at pH 3.5-4.0 in dilute (1:10) McLlvaine buffer, is recommended. The final preparation contrasts in color and transparency with the black, opaque silver grains.     

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.