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.     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

Metal Chelates as Reversible Stains for Detection of Electroblotted Proteins: Application to Protein Microsequencing and Immunoblotting

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.     

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   

Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase.

The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton.     

ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae

Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium. The cell walls accounted only for a small fraction of the cation retention by whole cells. ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm. ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins. The binding sites were formed by an amide and a strongly complexing ligand such as an amine. Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.     

Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S.

A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue.     

High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells

Scatchard analysis of binding of 125I-basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

Background  

Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.     

Decomposition of Sudan Black B Causing an Artefact in the Staining of Lipids

Sudan black B was found to be decomposed in acid solutions. This decomposition was rapid below pH 4. Above this pH the decomposition was markedly slower. After mild acid hydrolysis, the product was studied chromatographically and found to contain a basic dye with a pK of about 4, a brown color above, and a red color below pH 4. The nature of the product of hydrolysis indicates that it can stain nucleoproteins and mucopolysaccharides, hence a brownish stain is more indicative of artefact than the staining of lipids.     

Localization of methylene blue paramolybdate in vitally stained nerves

Methylene blue taken up by living neurons can be preserved for electron microscopy in a fixative containing osmium tetroxide and ammonium paramolybdate at pH 5.2. Paramolybdate is the buffer, precipitating agent and main osmotic ingredient; it does not function as an electron stain unless methylene blue is present. The low pH keeps the dye/paramolybdate complex from dissolving. Neither the low pH nor drastic dehydration from water to absolute ethanol harm the tissue. The staining mechanism involves cationic methylene blue associating with anionic structures such as microtubules and neurofilaments in the living cell; during fixation paramolybdate forms a precipitate with the dye at the staining sites. This fixative does not preserve microtubules unless they are first vitally stained.     

pH dependence and stoichiometry of binding to the Fc region of IgG by the herpes simplex virus Fc receptor gE-gI

Herpes simplex virus type 1 encodes two glycoproteins, gE and gI, that form a heterodimer on the surface of virions and infected cells. The gE-gI heterodimer has been implicated in cell-to-cell spread of virus and is a receptor for the Fc fragment of IgG. Previous studies localized the gE-gI-binding site on human IgG to a region near the interface between the C(H)2 and C(H)3 domains of Fc, which also serves as the binding site for bacterial and mammalian Fc receptors. Although there are two potential gE-gI-binding sites per Fc homodimer, only one gE-gI heterodimer binds per IgG in gel filtration experiments. Here we report production of recombinant human Fc molecules that contain zero, one, or two potential gE-gI-binding sites and use them in analytical ultracentrifugation experiments to show that two gE-gI heterodimers can bind to each Fc. Further characterization of the gE-gI interaction with Fc reveals a sharp pH dependence of binding, with K(D) values of approximately 340 and approximately 930 nm for the first and second binding events, respectively, at the slightly basic pH of the cell surface (pH 7.4), but undetectable binding at pH 6.0. This strongly pH-dependent interaction suggests a physiological role for gE-gI dissociation from IgG within acidic intracellular compartments, consistent with a mechanism whereby herpes simplex virus promotes intracellular degradation of anti-viral antibodies.     

Localization of membrane-associated sialomucin on the free surface of mesothelial cells of the pleura, pericardium, and peritoneum

 Strong anionic sites, as recognized by deposition of cationic colloidal iron even at pH 1.5, were distributed on the free surfaces of the mesothelia of the mouse pleura, pericardium, and peritoneum. Methylation inhibited colloidal iron staining on the surface, and successive saponification restored it. Digestion with neuraminidase or hydrolysis of sialic acid with H₂SO₄ erased the colloidal iron staining. Lectin Limax flavus agglutinin (LFA), which is specific for sialic acid, labeled the free surface of the mesothelium. All these findings strongly suggested that the surface substance contained sialic acid. Moreover, prior treatment with LFA inhibited the mesothelial surface stain with colloidal iron. In transmission electron microscopy, the colloidal iron (pH 7.3)-stained substance took the shape of fine strands of 50–300 nm in length. These characteristics of the substance on the mesothelial surface correspond well with biochemical properties of membrane-associated sialomucin, whose strong and abundant negative charges produce repulsive forces between facing serosal surfaces. This may contribute to prevent serosal adhesion and to reduce friction during movements of organs. Accepted: 7 January 1997     

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.     

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.     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites.

The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.