Luxol Fast Blue Arn: A New Solvent Azo Dye with Improved Staining Qualities for Myelin and Phospholipids

Luxol fast blue ARN (Du Pont, C.I. solvent blue 37) is a diarylguanidine salt of a sulfonated azo dye. This dye was compared with other Luxol blue and Luxol black dyes. Luxol fast blue ARN has improved staining qualities for phospholipids and myelin, and can advantageously be substituted for Luxol fast blue MBS (MBSN). Appropriate staining times for a 0.1% dye solution in 95% ethanol (containing 0.02% acetic add) at 35°-40° C range from 2-3 hr. After staining, the sections should be rinsed in 95% ethanol, rinsed in distilled water, and differentiated for 2 sec in 0.005% Li₂CO₃, rinsed in 70% ethanol, washed in water, and counterstained as required. Phospholipids and myelin selectively stain deep blue. A fixative containing CaCl₂, 1%; cetyltrimethylammonium bromide, 0.5%; and formaldehyde, 10%, in water gave excellent results with brain. However, 10% formalin can be used. The staining of the phospholipids is probably due to the formation of dye-phospholipid complexes.     

HNO3/H3PO4–NANO2 mediated oxidation of cellulose — preparation and characterization of bioabsorbable oxidized celluloses in high yields and with different levels of oxidation

The reaction of cellulose with a mixture of HNO₃/H₃PO₄–NaNO₂ (2:1:1.4, v/v/%w) at room temperature for different time intervals has been investigated to produce oxidized cellulose (OC), a biocompatible and bioresorbable polymer. The results revealed an increase in carboxyl content of OC with increasing reaction time, corresponding to about 8.0, 13.4, 17.4 and 18.4% carboxyl content after 12, 24, 36, and 48 h, respectively. The yield of OC ranged between 75 and 81%. The use of different ratios of HNO₃ and H₃PO₄, (11:1, 4:1, 2:1, 1:1, 1:2, and 1:4; v/v), in the reaction had no significant effect on the carboxyl content and yield of the OC products. All products, as produced, were low crystallinity (27–35%) fibrous materials. The length of fibers decreased with increasing reaction time. After ball milling for 24 h, the length of fibers further decreased and products converted into a fine powder consisting of small fibers and aggregated non-fibrous particles. The degrees of polymerization (DP) of the OC products produced after 12, 24, and 48 h of reaction duration were 81, 63, and 53, respectively. After ball milling for 24 h, the corresponding values changed to 57, 51 and 46. However, no significant change in the crystallinity of the products was noted after ball milling. The TGA results showed the OC products to be less thermally stable than cellulose. The degradation temperature appears to decrease with increasing carboxyl content. In conclusion, the results show that the low crystallinity OC products can be successfully prepared in high yields and with different levels of carboxyl content from cellulose by treatment with a mixture of HNO₃/H₃PO₄–NaNO₂.     

Horseradish peroxidase-catalyzed oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols

The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H₂O₂ as oxidative agent. The findings indicate that enzyme (H₂O₂ oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compounds. On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the observed increase in the absorbance at 502 nm (8,355 cm^{−1 −1} of extinction molar coefficient) due to the formation of a red azo dye compound resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system, HRP can be determined in picomolar levels by a fixed time method.     

Methylene Violet (Bernthsen), by Zinc-Alkali-Chlorate Hydrolysis of Methylene Blue

Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag₂O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na₂SO₃ of azures in CHCI₃, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO₃ (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO₃ in excess of the amount needed to convert the NaOH to Na₂CO₃. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC1₃ and extracting. The acetic layer should then be almost colorless while the CHC1₃ is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.     

Supravital Staining and Fixation of Brain and Spinal Cord by Intravascular Perfusion

Forty single and 13 combinations of dyes were tested for concomitant supravital staining and fixation of brain and spinal cord of rats, cats and squirrel monkeys by intravascular perfusion in 3 steps: (1) 60 or 80 ml of physiological saline containing 40 mg/100 ml of NaNO₂ as a vasodilator; (2) 250 or 550 ml of stain-fixative solution consisting of either: A—2 parts of dye solution in a concentration from 0.001% to 0.05% dissolved in saline containing 40 mg/100 ml of NaNO₂ and 1 part undiluted formalin; or B—2 parts of dye solution in a concentration from 0.001% to 0.05% dissolved in distilled water acidified with 1.5 ml of glacial acetic acid per 100 ml of water, and 1 part undiluted formalin; and (3) 100 or 150 ml of 6% dextrose in distilled water. Complete staining and fixation was accomplished in 53 min for rats and 41 min for cats and monkeys. Brains and spinal cord were frozen sectioned, and the cut surfaces of the frozen tissue were photographed similar to the procedure described by Gasteiger et al     

The Histochemical Detection of Thallium

Thallium can be histochemically localized in formalin-fixed, paraffin-processed tissues by treating sections, after passing them to water through xylene and graded alcohols, in a 0.5% aqueous Bi(NO₃)₃ solution acidified with 1 drop of concentrated HNO₃ per 100 ml. Sections are then washed and placed for 5 min in 2% aqueous KI, again washed, routinely dehydrated, cleared and covered. If desired, they may be lightly counterstained prior to dehydration. The tissue sites of T1 will be demonstrated by the fine wine-red crystals of (T1I)₂-BiI₃ resulting from the reaction, and will appear black with the usual microscopic illumination.     

Preparation of Tungsten Microneedles with A Propane Torch

Tungsten microneedless are easily fabricated from .005' to .010' diameter tungsten wire by exposing the free end of the wire to the blue inner cone of a fine-pointed flame from a propane gas torch. The needle is completed when the tip glows white hot and appears to shorten. The blue coating that results in biologically harmless but may be removed by dipping the needle briefly into fused NaNO₂ or Na₂SOs₃. Needles prepared in this way, and attached to suitable handles, are useful instruments in the fields of experimental embryology, tissue culture and electron microscopy.     

A Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however, the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl₂ · 6H₂O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

硒对亚硝酸钠导致的草鱼肝细胞氧化损伤的保护作用

以草鱼(Ctenopharyngodon idella)肝细胞(L8824)为研究对象,设置对照组、亚硝酸钠暴露实验组、亚硒酸钠孵育实验组和亚硒酸钠孵育后亚硝酸钠暴露实验组,探讨亚硒酸钠对不同浓度亚硝酸钠诱导L8824细胞氧化损伤及凋亡的保护作用。结果显示,亚硝酸钠暴露能抑制L8824细胞贴壁,导致细胞凋亡率增加。亚硝酸钠暴露引致L8824细胞的谷胱甘肽过氧化物酶(GPX)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性降低;gpx、sod和cat基因表达下调(P<0.05), DNA损伤诱导转录物3(ddit3)和bcl-2相关X蛋白(bax)基因表达上调(P<0.05)。亚硒酸钠(10μmol/L)孵育L8824对细胞形态和凋亡率无显著影响,但GPX、SOD和CAT活性上升,gpx、sod、cat、核因子E2相关因子2(Nrf2)和Kelch样环氧氯丙烷相关蛋白-1(keap1)基因表达上调(P<0.05)。亚硒酸钠孵育后亚硝酸钠暴露实验组,细胞凋亡率、GPX、SOD和CAT活性较对照组无显著变化,但B淋巴细胞瘤-2(bcl-2)基因表达显著上调(P<0...     

Phytoavailability assessment of heavy metals in soils by single extractions and accumulation by Phaseolus vulgaris

In Western Europe, policy makers are currently moving towards a more integrated risk-based approach of soil contamination assessment. As part of this approach, selective single extraction procedures have been proposed to add complementary insights regarding heavy metal behaviour and phytoavailability in soils and sediments. However, there is currently a wide range of such procedures available in literature, hampering standardisation and harmonisation of phytoavailability research of heavy metals. The current study examines shoot accumulation of Cd, Cu, Ni, Pb and Zn by the test plant Phaseolus vulgaris in 21 soils, differing in soil composition and level of contamination. On these soils, 12 different commonly used extraction procedures have been compared: soil solution extraction by Rhizon soil moisture samplers, 0.01 M CaCl₂, 0.1 M NaNO₃, 1 M NH₄NO₃, 1 M NH₄NOAc, 1 M MgCl₂, 0.11 M HOAc, 0.5 M HNO₃, 0.1 M HCl, DTPA–TEA–CaCl₂, EDTA-NH₄OAc and aqua regia. The plant species used in this study has previously been proposed as a test plant in a bioassay for assessing heavy metal induced oxidative stress in contaminated soils [Van Assche, F., Clijsters, H., 1990. A biological test system for the evaluation of the phytotoxicity of metal-contaminated soils. Environ. Pollut., 66, 157–172]. Cadmium shoot accumulation correlated best with soil solution concentrations, unbuffered nitrate solutions and the dilute CaCl₂ extraction procedure. The same was observed for Zn, yet for this element NH₄OAc and MgCl₂ also provided significant interactions. The best prediction for Ni was observed in the cluster containing CaCl₂ and NH₄NO₃. For Cd, Zn and Ni, the pseudo-total content and the aggressive chelate based and/or acidic extractants did not correlate well with shoot accumulation. Cu and Pb uptake on the other hand was found to correlate significantly (p = 0.01) with total content as well as with all aggressive extraction procedures over the range of soils used in this experiment. In general, the 0.01 M CaCl₂ extraction procedure proved to be the most versatile as it provided a good indication of phytoavailability for all five metals under evaluation.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

Sudan Black and Osmic Acid as Staining Agents for Testicular Interstitial Cells

Two standard cytological techniques have heen modified to stain specifically the interstitial cells of the testis. In Method 1, the tissue is fixed in Zenker-formol or Regaud's fluid for several hours or overnight and subsequently postchromed in 3% K₂Cr₂O₇ for 72 hr at 37°C. After paraffin embedding, sections are cut at 5μ, dewaxed, brought down to 70% alcohol and stained in an unfiltered saturated solution of Sudan black in 70% alcohol for 10-30 min. Sections are washed briefly in 70% alcohol to remove all excess dye, differentiated, if necessary, in 50% alcohol, downgraded to water and mounted in Farrants' medium or glycerol jelly. Interstitial cells: deep blue black; remainder of testicular tissue: light blue. Method 2 is essentially the Champy-Kull technique but specific staining for mitochondria is omitted and the sections are downgraded to water; then they are mounted in Farrants' medium or glycerol jelly without further treatment. In this way osmicated lipoids are preserved. Interstitial cells: conspicuous due to the variable number of black granules in their cytoplasm; the remainder of the tissue: yellow.     

光强和氮源及其浓度对缺刻缘绿藻生长、油脂和花生四烯酸积累的影响

对缺刻缘绿藻（Parietochloris incisa（Reisigl） S.Watanabe）在不同光强和氮源及其浓度条件下的生长状况及油脂和花生四烯酸（AA）的积累规律进行了研究。结果显示,缺刻缘绿藻在3种氮源条件下均能较好地生长。在高氮浓度条件下,增大光强能显著提高缺刻缘绿藻的生物量并促进油脂和AA的积累。缺刻缘绿藻在300 μmol·m^-2·s^-1光强、8.8 mmol·L^-1NaNO₃条件下生物量达到最大（4.17 g·L^-1）。油脂含量在100 μmol·m^-2·s^-1光强、1.0 mmol·L^-1氮浓度下达到最高,分别为41.17%（NaNO₃）、42.04%（NH₄HCO₃）和39.96%（CO（NH₂）₂）。AA绝对含量在300 μmol·m^-2·s^-1光强、2.9 mmol·L^-1 NaNO₃条件下达到最高,占细胞干重的16.44%。油脂和AA产率,在300 μmol·m^-2·s^-1光强、以NaNO₃为氮源的条件下达到最大,分别为134.6 mg·L^-1·d^-1（1.0 mmol·L^-1）和35.85 mg·L^-1·d^-1（2.9 mmol·L^-1）。综合考虑成本等因素,选择NH₄HCO₃（5.9 mmol·L^-1）和CO（NH₂）₂（2.9 mmol·L^-1）为氮源、在300 μmol·m^-2·s^-1高光强下培养缺刻缘绿藻进行AA的生产为最优方案。     

Oxazine Dyes. 2. Celestine Blue B, Gallocyanin and Gallamin Blue with Mordants Other Than Ferric Alum

Experiments with 3 oxazine dyes with 19 mordants failed to produce a more satisfactory staining solution than that recorded by Gray et al. (1956). The most practical solution developed is prepared by adding 0.4 ml concentrated HNO₃ to 1gm celestine blue B and dissolving the resulting mass in 100 ml 5% cupric nitrate containing 14 ml glycerol. This solution is less acid (pH 1.4) than the celestine blue B-ferric alum solution (pH 0.8) previously recommended. It gives as intense and sharp a stain but is slightly less stable. Formulae are given for four other combinations of possible practical application.     

The Identification of Some Acid Disazo Dyes by Paper Electrophoresis of their Reduction Products

Trypan blue, Niagara blue 2B, Niagara blue 4B, Afridol blue, Evans blue, Niagara sky blue 6B, Niagara blue 5B and Chlorazol blue G cannot be distinguished by their colour or their behaviour on paper chromatography. They may, however, be identified by paper electrophoresis at pH 4 of the products obtained by reduction of the azo linkages with sodium dithionite (Na₂S₂O₄).     

不同无机氮源对东海原甲藻生长的影响

在实验室条件下,研究了不同浓度、不同形态的氮(NaNO₃、NH₄Cl和NaNO₂)对东海原甲藻(Prorocentrum donghaiense)生长的影响。结果表明:在NH₄Cl浓度为5.20μmol·L^-1(N/P为8)时藻的比生长率最高,而N/P为32和100时,藻的生长明显受到抑制。在NaNO₃为氮源时,最适N/P为12(氮浓度为7.80μmol·L^-1)。而NaNO₂作氮源,N/P为16(10.40μmol·L^-1)时藻的比生长率最高,N/P为32和100时藻的生长也明显受到抑制。研究显示,东海原甲藻对无机氮NH₄Cl和NaNO₃和NaNO₂都可以利用,最适生长的N/P比范围在8～20之间,相对高的N/P(32、100)不利于东海原甲藻的生长。     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

Orthochromatic, Species-Limited Staining of Mast Cells With Night Blue

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

淹水条件下平邑甜茶根系NO生成及外源硝酸钠对其影响

以2年生平邑甜茶(Malus hupehensis)为试验材料,研究了淹水条件下平邑甜茶根系一氧化氮(NO)的生成规律以及外源硝酸钠(NaNO₃)对NO生物合成的影响.结果表明：3～9 d的淹水处理显著提高了平邑甜茶根系NO生成量;在12 d内,随着淹水时间延长,根系NO生成量、硝酸还原酶(NR)、一氧化氮合酶(NOS)活性和丙二醛(MDA)含量均先上升后下降.10 mmol·L^-1NaNO₃显著抑制了淹水条件下根系MDA含量和NOS活性的提高,但NR活性增强;淹水期间,根系NO生成量在NaNO₃处理的前3天提高,处理第6天后显著降低.     

Nitrite reduces the effects of HOCl on unsaturated phosphatidylcholines--a MALDI-TOF MS study

The concentration of nitrite (NO₂⁻) increases under inflammatory conditions. However, the physiological role of nitrite is so far controversial discussed: it was reported that effects of HOCl (an important inflammation mediator) on phospholipids (PL) may be enhanced but also reduced in the presence of nitrite.

In this paper a simple model system was used: unsaturated phosphatidylcholine (PC) vesicles were treated with HOCl in the presence of varying NaNO₂ concentrations and the yield of reaction products was determined by MALDI-TOF MS: the extent of chlorohydrin generation was significantly reduced in the presence of NaNO₂ because HOCl is consumed by the oxidation of NO₂⁻ to NO₃⁻.

Similar results were obtained when HOCl was generated by the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system or the experiments were carried out in the presence of a simple peptide. It is concluded that the transient products of the reaction between HOCl and NO₂⁻ do not have a sufficient reactivity to modify PL.