Procion brilliant orange, a counterstain for decalcified sections vitally labeled with procion brilliant red H-8 bs.

Procion brilliant red H-8 BS is a fluorescent dye that stains the organic matrix of bone supravitally. A procedure is described for counterstaining sections of such bone for visible light examination without interfering with the demonstration of sites of bound dye under UV illumination. Sections are brought to water, stained in Delafield's alum hematoxylin for 10 minutes, washed in tap water for 10 minutes, counterstained in 1% Procion brillant orange M-GS for 15 minutes and washed in distilled water for 10 minutes. After dehydration the sections were mounted in Eukitt.     

Marking of Olfactory Axons of Fishes by Intravital Staining with Procion Brilliant Yellow

Olfactory axons of fishes can be traced after vital staining of the olfactory surface with the fluorescent dye Procion brilliant yellow M 4-RAN. Following selective accumulation by the receptor dendrites and perikarya the dye migrates intra-axonally over distances of more than 200 μm. Fluorescence microscope observation of formaldehyde fixed and paraffin sectioned preparations of the olfactory organ demonstrate clearly the system of intraepithelial axons and their first order bundles running toward the olfactory nerve.     

Simple Fluorescence Staining of Insect Central Nerve Fibres with Procion Yellow

Nerve fibres in the central nervous system of the cockroach Periplaneta ameri-cana can be displayed by staining whole ganglia for 1-2 hr in a saturated solution of Procion yellow M-4R in cockroach saline diluted to maintain isotonicity. Selected fibres are stained preferentially by cutting a peripheral nerve or interganglionic connective close to the ganglion, or damaging neuron cell-bodies. The ganglion is washed in saline, fixed in alcoholic Bouin, dehydrated and embedded. Under fluorescence microscopy, sections show stained fibres brilliant yellow against a green background. The method is simpler than intracellular injection and demonstrates even the finest fibres.     

Cell-to-Cell diffusion of Procion Yellow in sheep and calf Purkinje fibers

Summary Sheep and calf Purkinje fibers (false tendons) were cut near one end and exposed to a solution containing no calcium and the dye Procion Yellow (M4RS, molecular weight near 700). Fifteen minutes later the damaged end was sealed by applying calcium ions (Tyrode solution). Traces of Procion Yellow were detected within the intracellular compartment at a distance of 2.4 mm from the site of damage when the preparations had been washed in dye-free solution for 4 hr. This indicates that the dye had diffused through about 20 cells in succession. There was no detectable uptake of Procion Yellow through intact surface membranes. Visual curve fitting to quantitative data on concentrationvs. distance gives an apparent diffusion coefficient (cell junctions and myoplasm in series) of 3×10^–8 cm² sec^–1, as against 1×10^–6 cm² sec^–1 in an agar gel. It is concluded that specialized contact areas between neighboring cardiac cells represent a considerable yet not an absolute hindrance to the movement of this particle.     

The interaction of yeast hexokinase with Procion Green H-4G.

1. A number of reactive triazine dyes specifically and irreversibly inactive yeast hexokinase at pH 8.5 and 33 degrees C. Under these conditions, the enzyme is readily inactivated by 100 microM-Procion Green H-4G, Blue H-B, Turquoise H-7G and Turquoise H-A, is less readily inactivated by Procion Brown H-2G. Green HE-4BD, Red HE-3B and Yellow H-5G and is not inactivated at all by Procion Yellow H-A. 2. The inactivation of hexokinase by Procion Green H-4G is competitively inhibited by the adenine nucleotides ATP and ADP and the sugar substrates D-glucose, D-mannose and D-fructose but not by nonsubstrates such as D-arabinose and D-galactose. 3. Quantitatively inhibited hexokinase contains approx. 1 mol of dye per mol of monomer of mol.wt. 51000. The inhibition is irreversible and activity cannot be recovered on incubation with high concentration (20 mM) of ATP or D-glucose. 4. Mg2+ protects the enzyme against inactivation by Procion Green H-4G but enhances the rate of inactivation by all the other Procion dyes tested. In the presence of 10 mM-Mg2+ the apparent dissociation constant between enzyme and dye is reduced from 199.0 microM to 41.6 microM. Binding of the dye to hexokinase is accompanied by characteristic spectral changes in the range 560-700 nm. 5. Mg2+ promotes binding of yeast hexokinase to agarose-immobilized Procion Green H-4G but not to the other dyes tested. Elution could be effected by omission of Mg2+ from the column irrigants or by inclusion of MgATP or D-glucose, but not by D-galactose. These effects can be exploited to purify hexokinase from crude yeast extracts. 6. The specific active-site-directed binding of triazine dyes to yeast hexokinase is interpreted in terms of the crystallographic structure of the hexokinase monomer.     

Vital Staining by Procion Navy Blue M3rs in Laboratory Mammals

Procion navy blue M3RS, a reactive textile dye for cotton, was given orally and parenterally to rats, rabbits, and dogs in dosages ranging from 50 to 150 mg/kg body weight. With intravenous injection, the animals became bright blue almost instantly. Dense staining of connective tissues, which occurred within minutes, persisted to variable degrees for several months. All connective tissue structures and mucin-containing structures were stained but no cellular or intracellular staining was seen. The dye has instantaneous anticoagulant activity which can be reversed. An in vitro dye concentration of 2 mg/ml of plasma prolongs clotting time to infinity, apparently by interfering with polymerization of fibrinogen. Even though many of the animals had nonclottable blood, they showed no hemorrhagic manifestations and most of the animals showed no other evidence of acute or chronic intoxication of consequence. Procion blue may be useful in the study of collagen and other connective tissues; since it contains copper, it should be sufficiently electron-dense to make it a useful in vivo stain for electron microscopy.     

Purification of hydrogenases by affinity chromatography on Procion Red-agarose.

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.     

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.     

Effect of Heat on the Antimicrobial Activity of Brilliant Green Dye

Antimicrobial activity of brilliant green dye in Trypticase soy broth (BBL) is reduced and ultimately destroyed by prolonged autoclaving at 121 C. Loss of antimicrobial activity is accompanied by decolorization of the dye. This is consistent with other evidence that antimicrobial activity of brilliant green resides in the colored dye ion. The dye is not decolorized when heated in distilled water or peptone, but is decolorized by heating in glucose, glycine, or sodium dodecyl sulfate, showing that decolorization results from reaction with components of the medium. To ensure optimal results, it is recommended that bacteriological media be sterilized by heat prior to addition of brilliant green dye.     

Affinity chromatography of acyl-CoA utilizing enzymes on Procion Red-agarose

The dye Procion Red HE3B immobilized on agarose and available as Matrex Gel Red A is shown to bind citrate synthase and succinate thiokinase from a number of diverse organisms. Saltgradient elution removes the enzymes in high yields and with substantial purification. The elution profiles follow a pattern similar to that of the molecular size variations of the enzymes.     

Specific inhibition of L-type pyruvate kinase by the triazine dye Procion Blue MX-R.

Incubation of the triazine dye Procion Blue MX-R with L- and M-type pyruvate kinase resulted in rapid time- and dye-concentration-dependent loss of activity. L-type pyruvate kinase was protected only by a low concentration of Mg2+; this was not the case with the M-type enzyme. Modification of the L-type form resulted in the incorporation of 1.54 +/- 0.057 mol of dye/mol of enzyme subunit in the absence of Mg2+, but only 0.73 +/- 0.024 mol of dye/mol of enzyme subunit in the presence of Mg2+. Tryptic peptide mapping of L-type pyruvate kinase modified in the presence and in the absence of Mg2+ further indicated that there were two sites modified in the enzyme, one of which was protected by Mg2+. The pKa of the nucleophile involved in the modification was calculated to be 7.1, implicating the possible involvement of a histidine residue. L-type enzyme was bound to Sepharose-immobilized Procion Blue MX-R specifically in the presence of Mg2+, whereas binding of the M-type enzyme was Mg2+-independent. The specific interaction of L-type pyruvate kinase with the dye was exploited in the large-scale purification of the enzyme and in the isolation of the phosphorylated enzyme.     

Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose

Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.     

Optimized dyeing conditions of immunoprotein with reactive dye Procion Blue MX-7RX

For investigating the feasibility of using reactive dyes as an immunoassay marker, a dichlorine triazine dye, Procion Blue MX-7RX, was employed to stain the antibody against human serum albumin (anti-HSA). With the color intensity revealed in the immunochromatographic test strip as the objective variables, the optimal dyeing conditions were found as follows: pH 11.4, temperature 35.7 degrees C, molar ratio 188 (mol dye/mol antibody), and reaction time 45.6 min. The dyed-anti-HSA revealed a maximal color intensity of 8738 without apparent loss of antigen binding affinity.     

Brilliant Green Bile Media

An attempt is made to determine the optimum concentration of bile and brilliant green in brilliant green lactose peptone bile medium, for use in the presumptive test in the bacteriological examination of water. Comparisons, showing the effect of bile and brilliant green upon the development of the colon organism, were made by making actual bacterial counts after a definite short incubation period. The results indicate that the original medium described by Muer and Harris, (5% dried bile, 1% peptone, 1% lactose, and 1:10,000 billiant green) when adjusted to pH = 7.1, did not show an appreciable inhibition of the colon organism. It is further shown that a medium containing 2% bile, 1% peptone, 1% lactose and 1:75,000 brilliant green supports a more rapid development of the colon organism at pH = 6.9 than does the original brilliant green bile medium at its optimum pH.     

Brilliant Green Bile Media

An attempt is made to determine the optimum concentration of bile and brilliant green in brilliant green lactose peptone bile medium, for use in the presumptive test in the bacteriological examination of water. Comparisons, showing the effect of bile and brilliant green upon the development of the colon organism, were made by making actual bacterial counts after a definite short incubation period. The results indicate that the original medium described by Muer and Harris, (5% dried bile, 1% peptone, 1% lactose, and 1:10,000 billiant green) when adjusted to pH = 7.1, did not show an appreciable inhibition of the colon organism. It is further shown that a medium containing 2% bile, 1% peptone, 1% lactose and 1:75,000 brilliant green supports a more rapid development of the colon organism at pH = 6.9 than does the original brilliant green bile medium at its optimum pH.     

Demonstration of Axial Elements of Sex Vesicles of Spermatocytes Using Coomassie Brilliant Blue

The axial element of sex chromosomes in the sex vesicle of rat and mouse spermatocytes has been visualized under the light microscope by the dye Coomassie brilliant blue (CBB). After staining in the CBB solution for 3-10 minutes, the axial elements appeared as darkly stained threads in the sex vesicles, whereas in controls stained with Giemsa or carbol fuchsin, the sex vesicles were usually uniformly stained. The axial elements are best seen when chromosome preparations were made by the flame drying technique. In rat spermatocytes the staining quality could be further improved by a brief treatment with trypsin solution (0.025%).

The CBB staining procedure is simple and easily controllable. The results suggest that the CBB stained material is protein in nature and is more resistant to trypsin digestion than other nuclear proteins.     

THE DISTRIBUTION OF SYNAPSES ON A PHYSIOLOGICALLY IDENTIFIED MOTOR NEURON IN THE CENTRAL NERVOUS SYSTEM OF THE LEECH : An Electron Microscope Study after the Injection of the Fluorescent Dye Procion Yellow

The fine structure of a physiologically identified motor neuron in the segmental ganglion of the leech central nervous system and the morphology of synapses on it were studied after injection of the fluorescent dye Procion yellow as a marker. The injected cell and its processes within the neuropil were located in thick or thin sections with fluorescence optics after initial fixation with glutaraldehyde and brief treatment with osmium tetroxide. The same or adjacent thin sections could then be examined in the electron microscope. Comparison with uninjected cells showed that the general features of the injected cell are retained although some organelles are distorted. The main features of the geometry of this neuron are the same from animal to animal: a single large process runs from the soma through the neuropil to bifurcate and enter the contralateral roots. Within the neuropil the main process gives off long branches (up to 150 µ), but these are greatly outnumbered by short branches and spines, one or a few microns in length, which were not appreciated in previous light microscope studies after injection of Procion yellow. Serial thin sections of selected areas along the main process within the neuropil showed that there are synapses on most of the shorter branches and spines; occasional synaptic contacts were also made on the main process itself and on longer branches. At least two morphologically distinct types of synapse could be recognized. A minimum estimate of the total number of synapses on the motor cell is 300, based on their occurrence in reconstructed segments.     

A Urea Silver Nitrate Method for Nerve Fibers and Nerve Endings

A silver nitrate stain for nerve fibers and endings applicable to paraffin sections on the slide utilizes the properties of urea to accelerate the procedure and improve the specificity of the stain. After removal of the paraffin the sections are run through absolute, 95% and 80% alcohol and placed for 60-90 minutes at 50-60°C. in: 1% aqueous silver nitrate, 100 ml.; urea, 20-30 g.; 1g. mercuric cyanide and 1 g. picric acid in 100 ml. of distilled water, 1-3 drops. After the silver bath they are rinsed quickly in 2 changes of distilled water and reduced for 3-5 minutes at 25-30°C. in: water, 100 ml.; sodium sulfite, anhydrous, 10g.; hydroquinone, 1-2g.; urea, 20-30g. They are then washed thoroughly in 4-5 changes of distilled water, passed through graded alcohols into 80% alcohol and examined under the microscope. If nerve fibers are not distinct, the sections are returned to the same urea-silver-nitrate bath for 10-15 minutes, rinsed, reduced, washed and dehydrated as before. This process may be repeated until staining is adequate; then they are dehydrated, cleared, and mounted.

Nerve fibers show a color range from brown to black; nerve cells from yellow to brown; and the background, depending on the type of tissue and its fixation, from yellow to light brown.     

Silver Staining of Nerve Fibers in Cardiac Tissue

Fresh hearts of dog were perfused through the coronary vessels with 1000 ml. of fixative (chloral hydrate, 5 g. per 100 ml. of 70% ethyl alcohol) and blocks of tissue 2 × 5 mm. from epicardium to endocardium fixed 48 hours in the same fixative. The blocks were placed in 95% alcohol containing 0.3% addition of strong ammonia for 4 hours, followed by 2 changes of plain 95% alcohol of 1 hour each, then cleared and infiltrated with paraffin. Mounted sections 12-15 µ thick were incubated in 1% silver proteinate (obtained from Serumvertrieb, Marburg, Germany)² at 38° C. for 48 hours in the presence of 10 g. of 15 gauge copper wire per 200 ml. of solution. The slides were rinsed gently in 3 changes of distilled water for 2 minutes, 1 minute and 1 minute, respectively, and reduced in 1% hydroquinone and 5% sodium sulfite for 5 minutes. They were washed 5 minutes in tap water and 5 minutes in 2 changes of distilled water and toned 3-5 minutes in 0.25% gold chloride, rinsed in distilled water 10 seconds, reduced 10 seconds in 1 % oxalic acid, rinsed 1 minute, fixed in 5% sodium thiosulfate 5 minutes, washed in tap water through 3 changes, dehydrated, cleared and covered. All solutions were made with distilled water except where otherwise specified. The results gave good impregnation of fine nerve fibers without the usual confusing staining of reticular tissue.     

Iron gallein elastic method---a substitute for Verhoeff's elastic tissue stain.

A method for staining elastic fibers in formalin fixed, paraffin embedded sections is described. After deparaffinizing and dehydration, sections are stained for 30 minutes in a solution prepared by mixing equal parts of 1% gallein dissolved in ethylene glycol and absolute alcohol (1:4), and 1.16% aqueous ferric chloride in 1% hydrochloric acid. The sections are washed in water and then differentiated in 2% ferric chloride for 2 minutes. After washing in water, the sections are counterstained with a variant of Van Gieson's picric acid-acid fuchsin for 1 minute. The results are similar to Verhoeff's elastic stain with elastic fibers staining black. An advantage to this staining procedure is that visually controlled differentiation is not necessary.