Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in Whole-Mount Skeletons in Vitro

Cartilage and bone of the developing skeleton can be reliably differentiated in whole-mount preparations with toluidine blue-alizarin red S staining after FAA fixation. The recommended staining procedure is based chiefly on the use of newborn white and Swiss-Webster mice, 4-9 days postnatal, but was tested also on mice and rats 3-8 wk of age. Procedure: Sacrifice, skin, eviscerate, remove body fat, and place specimens in FAA (formalin, 1; acetic acid, 1; 70% alcohol, 8) for approximately 40 min. Stain in 0.06% toluidine blue made in 70% ethyl alcohol for 48 hr at room temperature. Use 20 volumes of stain solution to the estimated volume of the specimen. Destain soft tissues in 35% ethyl alcohol, 20 hr; 50%, 28 hr; and 70%, 8 hr. Counterstain in a freshly prepared 1% aqueous solution of KOH to which is added 2-3 drops of 0.1% alizarin red S per 100 ml of solution. Each day for 3 days, transfer the specimen to a fresh 1% KOH-alizarin mixture, or until the bones have reached the desired intensity of red and soft tissues have cleared. Rinse in water, and place in a 1:1 mixture of glycerol and ethyl alcohol for 1-2 hr, then transfer the specimen to fresh glycerol-alcohol for final clearing and storage. Older mice and rats require procedural modifications: (1) fixation for 2 hr, (2) 0.12% toluidine blue, (3) maceration for 4 days in 3% KOH-alizarin, and (4) preliminary clearing for 24 hr in a mixture of glycerol, 2; 70% ethyl alcohol, 2; and benzyl alcohol, 1 (v/v) before placing in a 1:1 alcohol-glycerol mixture.     

Structural studies on aqueous and hydroalcoholic solutions of a polyene antibiotic: Amphotericin B

Circular dichroism and absorption and light scattering have been used to study the effect of ethyl alcohol on an aqueous solution of Amphotericin B (“Fungizone”), which is an antifungal heptene. In aqueous solution, light-scattering studies show that Amphotericin B exists in an aggregated form. The estimated mass of these aggregates is about 2 × 10⁶ daltons, representing about 2000 molecules. Since the aggregated form is high molecular weight and scattering, the CD involves probably the differential scattering of right and left polarized light. In aqueous solution, Amphotericin B exhibits a strong dissymetric couplet in CD at the wavelength of the absorption maximum (328 nm). This latter maximum presents a blue shift when compared with the normal absorption in polar organic solvents. In hydroalcoholic solutions, for alcohol concentrations below 35%, the molecular weight of the aggregates is unchanged, while the absorption and CD spectra are modified. For alcohol concentrations greater than 35%, the aggregates mass decreases quickly and becomes undetectable at 50% ethyl alcohol concentration. For these solutions, the CD and absorption spectra are practically constant and characteristic of unaggregated Amphotericin B form.     

The Histological Examination of Mummified Material

Tissue from Egyptian mummy material is extremely brittle; hence it was handled in perforated glass tubes during processing. The first (softening) fluid consisted of 96% ethyl alcohol, 30 vol; 1% aqueous formalin, 50 vol; 5% aqueous Na₂CO₃, 20 vol. It was used in a fluid to tissue volume ratio of 100:1 and allowed to act overnight. A special dehydrating sequence: 80% alcohol, 3-6 hr; 8% phenol in 96% alcohol, and absolute alcohol followed by 3 changes of amyl acetate, 6-18 hr each; 3 changes of 1 % celloidin in methyl benzoate, 24 hr each; then through benzene and embedding in paraffin completed the special technic. This allowed regular sectioning and staining to be done successfully.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Determination of amlodipine using terbium‐sensitized luminescence in the presence of europium(III) as a co‐luminescence reagent

A sensitive time‐resolved luminescence method for the determination of amlodipine (AM) in methanol and in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb³⁺) by formation of a ternary complex with AM in the presence of tri‐n‐octylphosphine oxide (TOPO) as co‐ligand, dodecylbenzenesulfate as surfactant and europium ion as a co‐luminescence reagent. The signal for Tb–AM–TOPO is monitored at λ_ex = 242 nm and λ_em = 550 nm. Optimum conditions for the formation of the complex in aqueous system were 0.015 m Tris (hydroxylmethyl) amino methane buffer, pH 9.0, TOPO (1.0 × 10^–4 m ), Eu³⁺ (2.0 × 10^–7 m ), dodecylbenzenesulfate (0.14%) and 6.0 × 10^–5 m of Tb³⁺, which allows the determination of 10–50 ppb of AM with a limit of detection of 1.2 ppb. The relative standard deviations of the method range between 0.1 and 0.2% indicated excellent reproducibility of the method. The proposed method was successfully applied for the assay of AM in pharmaceutical formulations and in plasma samples. Average recoveries of 98.5 ± 0.2% and 95.2 ± 0.2% were obtained for AM in tablet and plasma samples respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Observing Pollen Tubes Within the Styles of Zea Mays L.

With the method herein described, pollen tubes of Zea mays L. could be observed within the style, from the exposed stigmatic surface to the base of the style. At different periods after pollination whole ears were fixed and stored in Karpechenko's modification of Navashin's solution. Silks were removed from the ears, dehydrated in an ethyl alcohol series up to 80%, and stored therein. The preparation of the slides was as follows: (1) 50% ethyl alcohol, 2 minutes (2) 15% ethyl alcohol, 2 minutes (3) boiling distilled water, 10 minutes (4) 1% potassium permanganate, 15 minutes for a 4 cm. portion of silk and 1 hour for a whole silk (5) 1% oxalic acid, long enough for the silk to turn white (6) 70% ethyl alcohol, 2 minutes (7) macerating solution (equal parts of concentrated HO and 95% ethyl alcohol), 1 hour (8) 70% ethyl alcohol, 2 minutes or stored until examination (9) lactophenol, 2 minutes (10) mounted in lactophenol and (11) squashed. The preparations were examined with a dark field microscope.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

Staining Paraffin Sections with Protargol 6. Impregnation and Differentiation of Nerve Fibers in Adrenal Glands of Mammals

A series of experiments with protargol staining of nerve fibers in mammalian adrenal glands has yielded the following procedure: Fix-1-2 days in a mixture of formamide (Eastman Kodak Company) 10 cc, chloral hydrate 5 g., and 50% ethyl alcohol 90 cc. Wash, dehydrate and embed in paraffin. Cut sections about 15 and mount on slides. Remove the paraffin and run down to distilled water. Mordant 1-2 days in a 1% aqueous solution of thallous (or lead) nitrate at 56-60°C. Wash thru several changes of distilled water and impregnate in 1% aqueous protargol (Winthrop Chemical Company) at 37-40°C. for 1 to 2 days. Rinse quickly in distilled water and differentiate 7-15 seconds in a 0.1% aqueous solution of oxalic acid. Rinse thru several changes of distilled water for a total time of 0.5 to 1.0 rain. Reduce 3-5 rain, in Bodian's reducer: hydroquinone 1 g., sodium sulfite 5 g., distilled water 100 cc. Wash in running water 3-5 min. and tone 5-10 min. in a 0.2% gold chloride solution. Wash 0.5 min. or more and reduce in a 2% oxalic acid solution to which has been added strong formalin, 1 cc. per 100. (Caution. This last reduction is critical and over-reduction can spoil an otherwise good stain; 15-30 seconds usually suffices, and the sections should show only the beginning of darkening to a purplish or gray color.) Wash, fix in hypo, wash, dehydrate and cover.     

Synthesis and characterization of Y(1‐x)Eu(x)(TTA)3(Phen) organic luminescent thin films

Yttrium is stoichiometrically doped into europium by mole percentage, during the synthesis of Y_(1‐x)Eu_(x)(TTA)₃(Phen), using solution techniques (where x = 0.2, 0.4, 0.5, 0.6 and 0.8, TTA = thenoyltrifluoroacetone and Phen = 1,10‐phenanthroline).These complexes were characterized using different techniques such as X‐ray diffraction, thermogravimetric/differential thermal analysis, optical absorption and emission spectra. Thin films of the doped Eu–Y complexes were prepared on a glass substrate under a high vacuum of 10^‐6 Torr. The photoluminescence spectra of these thin films were recorded by exciting the sample at a wavelength of 360 nm. The emission peak for all the synthesized complexes centered at 611 nm; maximum emission intensity was obtained from Y_0.6Eu_0.4 (TTA)₃(Phen). The results proved that these doped complexes are more economical than pure Eu(TTA)₃(Phen) and are best suited as red emissive material for energy‐efficient and eco‐friendly organic light‐emitting diodes and displays. Copyright © 2013 John Wiley & Sons, Ltd.     

Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.     

Some New Methods for Affixing Sections to Glass Slides. I. Aqueous Adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyl-triethoxysilane-either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions-was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

Some new methods for affixing sections to glass slides. I. Aqueous adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyltriethoxysilane--either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions--was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

Synthesis and characterization of novel europium β‐diketonate organic complexes for solid‐state lighting

Volatile Eu complexes, namely Eu(TTA)₃Phen, Eu_(x)Y_(1‐x)(TTA)₃ Phen; Eu_(x)Tb_(1‐x)(TTA)₃Phen; Eu, europium; Y, yttrium; Tb, Terbium; TTA, thenoyltrifluoroacetone; and Phen, 1,10 phenanthroline were synthesized by maintaining stichiometric ratio. Various characterization techniques such as X‐ray diffraction (XRD), photoluminescence (PL) and thermo gravimetric analysis/differential thermal analysis (TGA/DTA) were carried out for the synthesized complexes. Diffractograms of all the synthesized complexes showed well‐resolved peaks, which revealed that pure and doped organic Eu³⁺ complexes were crystalline in nature. Of all the synthesized complexes, Eu_0.5 Tb_0.5(TTA)₃Phen showed maximum peak intensity, while the angle of maximum peak intensity for all complexes was almost the same with slightly different d‐values. A prominent sharp red emission line was observed at 611 nm when excited with light at 370 nm. It was observed that the intensity of red emissions increased for doped europium complexes Eu_(x)Y_(1‐x)(TTA)₃Phen and Eu_(x)Tb_(1‐x)(TTA)₃ Phen, when compared with Eu complexes. Emission intensity increased in the following order: Eu(TTA)₃Phen > Eu_0.5 Tb_0.5(TTA)₃Phen > Eu_0.4 Tb_0.6(TTA)₃Phen > Eu_0.5Y_0.5(TTA)₃Phen > Eu_0.4Y_0.6(TTA)₃Phen, proving their potential application in organic light‐emitting diodes (OLEDs). TGA showed that Eu complexes doped in Y³⁺ and Tb³⁺ have better thermal stability than pure Eu complex. DTA analysis showed that the melting temperature of Eu(TTA)₃ Phen was lower than doped Eu complexes. These measurements infer that all complexes were highly stable and could be used as emissive materials for the fabrication of OLEDs. Copyright © 2012 John Wiley & Sons, Ltd.     

Time-resolved immunofluorometric detection of antigens separated by high-performance liquid chromatography and coated to polystyrene.

We use high-performance liquid chromatography with fraction collection to separate an antigen of interest. The antigen is then immobilized on polystyrene microtiter wells and detected with a specific antibody, followed by a second biotinylated antibody and streptavidin labeled with a fluorescent europium chelate. Fluorescence can be quantified with the use of time-resolved fluorescence. Antigen detectability down to 2-3 x 10(-17) mol was achieved. This method could be used as an alternative to Western blot in certain applications.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

Stereognostic coordination chemistry 4 the design and synthesis of a selective uranyl ion complexant

A new approach to ligand design for the sequestration of metal-oxo cations has been called stereognostic coordination chemistry, in that the ligand incorporates a traditional Lewis base coordination to the metal center and a hydrogen bond donor to interact with the oxo group. This paper reports the synthesis of ligands that are more rigid and sterically predisposed to bind the targeted UO₂²⁺ cation. These are the tripod ligands tris-N,N′,N′′-[2-(2-carboxy-phenoxy)ethyl]-1,4,7-triazacyclononane bis-hydrochloride (ETAC · 2HCl) and tris-N,N′,N′′-[2-(2-carboxy-4-decyl-phenoxy)ethyl]-1,4,7-triazacyclononane tris-hydrochloride (DETAC · 3HCl), which chelate uranyl with a tris-carboxylate coordination sphere and provide a hydrogen bond donor through a protonated amine on the triazacyclononane macrocycle to interact with one uranyl oxo atom. Structural models predict that upon uranyl binding the hydrogen bond donor must point directly towards the oxo atom, enforcing a stereognostic interaction. Both ETAC and DETAC chelate the uranyl ion; DETAC is a powerful extractant and will quantitatively extract uranyl into an organic phase at pH 1.9 and above. The extraction coefficient is estimated to be 10¹⁴ in neutral aqueous conditions. Vibrational spectra of ¹⁸O labeled UO₂²⁺ have been used to probe the stereognostic coordination to uranyl utilizing hydrogen bonding.     

Synthesis of a coumarin-based europium complex for bioanalyte labeling

A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved fluorescence measurements has been designed. Compound 1 displays three functional elements: an azide reactive spacer arm, a coumarin sensitizer, and a seven-coordinate europium complex. That complex can be excited at 370 nm by inexpensive UV-LEDs as a light excitation source.     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.