A HYDROXYL-DEPENDENT SILVER METHOD FOR NERVE AXOPLASM

Axoplasm is selectively impregnated by the following steps: (1) fixation in 10% formalin or in 10% formalin with added sucrose, 15%, and concentrated NH₄OH, 1%, for 1-7 days; (2) frozen sections; (3) extraction of the sections in 95% ethyl alcohol, absolute alcohol, xylene, and 95% ethyl alcohol and absolute alcohol, 1 hr each; (4) distilled water, 3 changes of 10 min each; (5) 20% AgNO₃ (aq.) at 25°C, 30 min; (6) distilled water, 3 changes of 1-2 sec each; (7) 6.9% K₂CO₃, 1 hr; (8) water, 3 changes of about 1 min each; (9) 0.2%AuCl₃, 2 min; (10) distilled water; (11) 5% Na₂S₂O₃, 2 min; (12) washing, clearing and mounting. This procedure is proposed as a simplified stain for axoplasm, with other tissue components remaining unstained. The few reagents necessary suit this method for histochemical investigation of the mechanism of silver staining.     

A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

温度和气体成分对冬枣果实贮藏期间品质的影响

主要探讨冬枣(Ziziphus jujuba Mill.cv Dongzao)在-1℃的动态气调(CA-Ⅰ,70%O2 0%CO2处理7天,然后转入5%O2 0%CO2中)、普通气调(CA-Ⅱ,5%O2 0%CO2;CA-Ⅲ,10%O2 0%CO2)及普通冷藏和常温(25℃)等条件下,果实发病率、色素、可溶性固形物、可滴定酸、乙醇和乙酸乙酯含量等的变化.结果表明:与普通冷藏相比,气调贮藏能减缓果实的腐烂,抑制色素的分解和减少果肉中乙醇、乙酸乙酯的含量.动态高氧处理能有效地保持果实的颜色,抑制色素降解及果皮褐变.CA-Ⅲ(10%O2 0%CO2)能有效地减少果肉乙醇的含量而CA-Ⅱ(5%O2 0%CO2)能有效地减少果肉乙酸乙酯的含量.气调贮藏的果实可滴定酸及可溶性固形物含量与其他处理的果实没有明显的差异.     

CHEMICAL STRUCTURAL REQUIREMENTS FOR INDUCTION OF OOCYTE MATURATION AND SPAWNING IN STAREISHES

Effects of various adenine derivatives on oocyte maturation and spawning were studied in the starfishes, Marthasterias glacialis, Astropecten aurantiacus, Patiria miniata, Asterina pectinifera and Asterias forbesi . 1-Methyladenine and 1-ethyladenine were very effective in inducing oocyte maturation and spawning, whereas the following related compounds had no effect: adenine, 3-methyladenine, 7-methyl-adenine, 9-methyladenine, 1-methylguanine, 1-methylhypoxanthine, 6-methylpurine, N₆-methyladenine, N₆-
dimethyladenine, N₆-benzyladenine, N₆-furfuryladenine(kinetin), adenosine, 5' -adenylic acid, adenosine 3',5'-cyclic monophosphate, adenosine triphos-phate, inosine, 5'-inocinic acid, guanine, guanosine, 5'-guanylic acid, hypoxanthine, xanthine, xanthosine, 3-methylcytidine and 5-methylcytosine. 1-Methyladenosine induced oocyte maturation and spawning when isolated ovarian fragments were used as assay material; however, it had little effect in inducing maturation of isolated oocytes. Therefore, this compound seems to active only after its decomposition to 1-methyladenine and ribose. The chemical structure responsible for inducing oocyte maturation and spawning in starfishes is proposed: a short alkyl radical such as methyl or ethyl at N₁ site and an imino radical at C₆ site of the purine nucleus.     

Biocatalytic, Enantioselective Preparations of (R)- and (S)-Ethyl 4-Chloro-3-Hydroxybutanoate, a Useful Chiral Synthon

The synthesis of optically active ethyl 4-chloro-3-X-butanoate derivatives la-d (X = OH, a; OCOCH₃, b; OCOC₃H₇, c; OCH₂C₆H₅, d) was realized using various biocatalytic approaches such as microbiological reduction of ethyl 4-chloro-3-oxobutanoate 2 with lactic acid bacteria, hydrolysis of lb-d by the hydrolytic enzymes PLE and BChE and the transesterification of la catalyzed by a lipase from Pseudomonas fluorescens (PFL).     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

Counterstaining Golgi-Cox Impregnations with Luxol Fast Blue as a Myelin Stain

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K₂Cr₂O₇, 1 gm; HgCl₂, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K₂CrO₄, 0.8 gm; Na₂WO₄, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO₃, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5-10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5-60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li₂CO₃ for 3-4 min. Differentiate in 70% ethanol and place in water. Immerse for 5-15 min in a mixture of two solutions: A. cresylechtviolet (Otto C. Watzka, Montreal), 2 gm; 1 M acetic acid, 185 ml; B. 1 M sodium acetate, 15 ml; distilled water, 400 ml; absolute ethanol, 200 ml. Dehydrate to 3:1 ethanol-chloroform. Clear in toluene and apply a coverslip. The technique produces fast Golgi-Cox impregnated neurons against a background of counterstained myelinated fibers. Patterns of the myelinated fibers can be used to localize impregnated neurons.     

蝉虫草样品的分级萃取与化学成分分析

利用索氏萃取技术,依次采用石油醚、乙酸乙酯、丙酮、无水乙醇和甲醇等5种溶剂对蝉虫草纯粉进行分级萃取,运用傅里叶变换红外光谱分析法和气相色谱-质谱联用技术对5级萃取物进行分析鉴定。傅里叶变换红外光谱分析显示萃取物中含有与烯烃类、羧酸类、酯类、醇类和酮类等化合物相关的C-H、C=O、C-O和C=C等官能团。气相色谱-质谱联用技术共鉴定出有机小分子化合物34种,以酯类和脂肪酸类为主,多为碳链长度为15-20的长链脂肪酸及对应的酯,其中十八碳不饱和脂肪酸相对含量高达28.95%;分别存在于两种或以上萃取物中的有机化合物共有11种;仅存于石油醚萃取物中的化合物6种,乙酸乙酯萃取物中3种,丙酮萃取物中2种,无水乙醇萃取物中6种,甲醇萃取物中6种。在一定极性范围内,利用溶剂的极性梯度变化,可实现蝉虫草中活性物质的按极性梯度分离;采用分级萃取技术可有效分离蝉虫草中部分化学成分。鉴定结果充实了蝉虫草中化合物的种类资源,为蝉虫草中活性物质谱图库的完善、构效关系的建立及蝉虫草产品的利用开发提供支撑。     

A Simple Histochemical Reaction for Aldehydes

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0-0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2-3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1-0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3-5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

Injection of Lymphatics: With Colored Cedar Oil; With Plastic

(1) The oil mass consists of: cedar oil, 1; color in oil (a paint pigment, e.g., Prussian blue), 1; and toluene, 2, parts by volume. To use, add 1 ml of diethyl ether to each 10 ml of mass, mix thoroughly and inject into the fresh organ with a very fine glass or metallic needle. Heat the organ in water at 50-60° C before starting the injection, massage gently after injection, then fix. For macroscopic studies, fix 5 days in 5% formalin, and dissect. For microscopic studies, fix at least 5 days in: formalin, 10 ml; Al₂(SO₄)₃, 2 gm; ZnSO₄, 2 gm; acetic acid, 4 ml; and distilled water, 90 ml. Dehydrate with dioxane, embed in paraffin and section at 10-20 μ. Stain with hematoxylin-eosin or with one of the following modifications of Van Gieson's formula: 1. 1% acid fuchsin, 10; picric acid (sat. aq.), 50; and 5% ZnSO₄, 40 volumes. 2. 1% acid fuchsin, 20; picric acid (sat. aq.), 80; and 5% CoSO₄, 40 volumes.

(2) The plastic mass consists of a 5-10% solution of Rhodopas (a vinyl copolymer) in acetone. Injection is made as with the oil mass except that a plastic squeeze-bottle and glass needle is preferable to a syringe. Indirect injection is used for both procedures, i.e., into the organ substance; not into a cannulated lymphatic vessel. After the plastic has hardened (24 hr), the unfixed tissue is subjected to corrosion by 5-10% NaOH in water.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

Marchi's Staining Method: Studies of Some of the Underlying Mechanisms Involved

Spinal cord of cat and rabbit was stained, after experimental lesions, by variations of Marchi's method. The following conclusions were drawn:

1. The presence of an oxidizing agent (K₂Cr₂O₇, NaIO₃, or KCIO₃) in the osmic acid solution is of primary importance and a preliminary oxidation in Mueller's fluid is unnecessary or even detrimental.

2. Acetic acid added to Marchi's fluid, accentuates the action of the oxidizing agent in restraining the staining of normal myelin.

3. Too high concentration of oxidizing agent or of acid may inhibit staining of degenerating myelin.

4. Marchi's and Busch's methods have been modified as follows: Fix one day in 10% formalin and transfer without washing to the staining mixture, either A or B. Staining mixture A: Marchi's fluid plus 1 to 3% glacial acetic acid. B: An aqueous solution containing KCIO₃ 0.25%, osmic acid 0.33%, and acetic acid 1%. Stain about one week. These methods worked on spinal cord and medulla, but cannot be recommended for brain.

5. The detrimental effects of long post mortem autolysis or of prolonged fixation in formalin may be counteracted to some degree by increasing the concentration of the acid in Marchi's fluid up to 5% or of the KCIO₃ up to 0.4% in the modified Busch's fluid.     

Tyrosine attack by free radicals derived from catalytic decomposition of carbon tetrachloride

The interaction between free radicals derived from the catalytic decomposition of carbon tetrachloride and tyrosine (the N-acetyl tyrosine ethyl ester, ATEE) under anaerobic and aerobic conditions was studied. The structure of the reaction products formed was desciphered by the GLC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions the formation of the following products was found: (1) an unsaturated derivative of the amino acid; (2) the trimethylsilyl derivative of N-acetyl chloro tyrosine ethyl ester; (3) a hydroxyl adduct of ATEE ; (4) an ATEE adduct having a chlorine and a CCl₃ group in the molecule (it is suggested that CCl₃ is attached to the benzyl carbon and the chlorine located in the benzene ring); (5) an ATEE adduct having only a CCl₃ group tentatively assigned to be located on the benzyl carbon; and (6) and (7) were found to be two isomers of an ATEE having one CCl₃ on the aromatic ring. Under aerobic conditions the following reaction products were identified: Two products which were similar to those numbered (1) and (2) and formed anaerobically; (8) and (11) two isomeric dichlorinated adducts of ATEE; (9) and (10) two isomeric dichlorinated monohydroxylated derivatives of ATEE. Concerning the potential relevance of these findings, we consider that if similar interactions to those here reported occurred during CCl₄ poisoning, the activity of enzymes having tyrosine in their active center might result in impairment. Further, enzymes operating on tyrosine moieties in proteins might be perturbed in their action if tyrosine groups were attacked by the free radicals arising from catalytic decomposition of CCl₄ evidenced here.     

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.     

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.     

Presence of glycerol teichoic acid in the cell wall of Lactobacillus kefiranofaciens

The accessory polymer of the capsular polysaccharide of a 'kefiran'-producing Lactobacillus kefiranofaciens was studied. The teichoic acid of L. kefiranofaciens K₁ was extracted from the cell with 5% trichloroacetic acid (w v) at 5°C for 24 h and was purified by ion-exchange chromatography on DEAE-Toyopearl 650 M with a linear gradient of (NH₄)₂CO₃; the yield was only 2%. The teichoic acid has a molecular weight of 22000 and is composed of D-glucose, 2-acetamide-2-deoxy-D-glucose, glycerol and phosphorus in a molar ratio of 1.0 : 2.0 : 2.3 : 1.1. The low yield of teichoic acid suggested that kefiran is the main accessory polymer in the cell-wall of L. kefiranofaciens.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Ruthenium complex catalyzed polymerization of OH or COOH group containing alkynes to give functionalized poly(acetylene)s

The ruthenium(III) complex [(Cp*)RuCl₂]₂ (Cp*=permethylcyclopentadienyl) catalyzes polymerization of propiolic acid to give a mixture of poly(propiolic acid), [---CH=C(COOH)---]_n (1), and cyclic trimers, 1,2,4- and 1,3,5- benzenetricarboxylic acids. GPC analysis shows MN and MW values of the polymer of 4.0 × 10³ and 4.3 × 10³, respectively. Reaction of propiolic acid in the presence of the Ru(II) complex, (Cp*)RuCI(L) (L=1,5-cyclooctadiene and norbornadiene), gives the cyclic trimers rather than 1. [(Cp*)RuCl₂]₂ catalyzes polymerization of acetylenedicarboxylic acid and of propargyl alcohol to give the corresponding poly(acetylene) derivatives, [---C(COOH)=C(COOH)---]_n (2) and [---CH=C(CH₂OH)---]_n (3), respectively. Polymerization of ethyl propiolate, 2-butyn-1,4-diol, phenylacetylene and (trimethylsilyl)acetylene using [(Cp*)RuCl₂]₂ gives the corresponding polymers [---CH=C(COOEt)---]_n (4), [---C(CH₂OH)=C(CH₂OH)---]_n (5), [---CH=CPh---]_n (6) and [---CH=C(SiMe₃)---]_n (7) in low yields.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).