Prolonged methanol fixation of soluble mucosubstances in mucopolysaccharidoses

Summary A simple and efficient method for the demonstration of highly water soluble acid mucosubstances in cold microtome sections is described. It consists of prolonged treatment of cold microtome sections with methanol (for at least 1 h) and subsequent staining with 0.1% azure A in distilled water or in 30% methanol. The procedure is recommended particularly for the bioptical examination of mucopolysaccharidoses.     

A modification of the tannic acid-phosphomolybdic acid-dye stain for demonstrating myoepithelial cells in formalin fixed tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin sections are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:1 methanol:acetic acid, rinsed in 9:1 methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Identifying decomposition products in extracts of cellular metabolites

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, then, it would seem that the best method for extracting a particular metabolite is the one that gives the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that >or=50% water gave an increased yield of nucleosides and bases compared with 相似文献   

Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum

Background

A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism.

Results

Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. Only the solutions containing either 70 mM HEPES or 0.85% (w/v) ammonium carbonate (pH 5.5) caused less than 10% cell leakage and the energy charge of the quenched cells was high, indicating rapid inactivation of the metabolism. The efficiency of extraction of intracellular metabolites from cell cultures depends on the extraction methods, and is expected to vary between micro-organisms. For L. plantarum, we have compared five different extraction methodologies based on (i) cold methanol, (ii) perchloric acid, (iii) boiling ethanol, (iv) chloroform/methanol (1:1) and (v) chloroform/water (1:1). Quantification of representative intracellular metabolites showed that the best extraction efficiencies were achieved with cold methanol, boiling ethanol and perchloric acid.

Conclusion

The ammonium carbonate solution was selected as the most suitable quenching buffer for metabolomics studies in L. plantarum because (i) leakage is minimal, (ii) the energy charge indicates good fixation of metabolism, and (iii) all components are easily removed during freeze-drying. A modified procedure based on cold methanol extraction combined good extractability with mild extraction conditions and high enzymatic inactivation. These features make the combination of these quenching and extraction protocols very suitable for metabolomics studies with L. plantarum.     

Viscous Product from Activated Sludge by Methanol Fermentation

Aeration of activated sludge with 3 to 4% added methanol for 5 to 7 days yields an odorless, highly viscous (5,000 to 10,000 centipoise), black, pudding-like product containing glycan(s) linked other than α-1-4 or β-1-3. Backseeding gives maximum thickening in 3 to 4 days. Incomplete acid hydrolysis of the black product gives a 0.27% solution of reducing sugars (75% glucose) which is an 11.4% yield from the added methanol. Backseeding into either centrifuge supernatant or 0.1% yeast extract in tap water gives a light-colored polymer. Viscosity decreases during extended sterile cold storage. A 5% salt addition lowers viscosity one-half. From 6 to 12 colony types appear on plating backseeded media, but none of these isolates is a reliable polymer former.     

Leakage of adenylates during cold methanol/glycerol quenching of Escherichia coli

Effective and rapid inactivation of cellular metabolism is a prerequisite for accurate metabolome analysis. Cold methanol quenching is commonly applied to stop any metabolic activity and, at the same time remaining the cells’ integrity. However, it is reported that especially prokaryotic cells like Escherichia coli and Corynebacterium glutamicum tend to leak intracellular metabolites during cold methanol quenching. In this work leakage of adenylates is quantified for different quenching fluids. Further, a methanol/glycerol based quenching fluid is proposed, which reduces leakage drastically compared to the commonly applied methanol/water solution (16% ATP leakage compared to more than 70%).     

A study of acid mucopolysaccharides in cold microtome sections of normal and experimentally hydrated bovine corneas

Summary Acid mucopolysaccharides were investigated in cold microtome sections of normal and experimentally hydrated bovine corneas. Staining methods using cationic dyes were used for the detection.A 10 min fixation of cold microtome sections in absolute alcohol did not change the stainability of acid mucopolysaccharides substantially. The staining was only a little fainter (as against unfixed sections). After 10 min fixation with formol-cetylpyridinium chloride the staining of sections was diminished and after 30 min fixation in this fluid completely abolished. After formol-calcium chloride fixative the staining was decreased in dependence on the time of fixation due to the elution of acid mucopolysaccharides in the fixative (acid mucopolysaccharides in the fixative were demonstrated by means of paper electrophoresis). Formolcalcium chloride is likewise unsuitable.Experimental hydration of corneas in distilled water did not substantially alter the staining properties of acid mucopolysaccharides in cold microtome sections. Only quantitative differences were found in comparison with untreated corneas. These differences were due to hydration causing an increase in the distance of acidic groups among individual molecules of acid mucopolysaccharides.     

The Use of Ba(OH)2 in Methanol for Demonstration of the Distribution of Sucrose in Sugarbeet Roots

Sucrose in the tissues of the sugarbeet (Beta vulgaris L.) can be shown as follows. Fresh or stored roots are cut into pieces having a block face of about 1 × 2 cm, and sections of about 150 μ thickness prepared from these. The sections are rinsed 15-30 sec in iced distilled water, placed in Ba(OH)₂-saturated methanol for 3 min and then rinsed twice in methanol for 1 and 5 min respectively. They are then transferred to ethanol through a graded series consisting of 80, 60, 40, and 20% methanol in ethanol, 5 min in each, with evacuation as necessary to remove bubbles. Temporary mounting is in ethanol and examination made by incident light or darkfield illumination. Gradual replacement of ethanol with xylene permits mounting in a resinous medium. An opaque granular precipitate of barium saccharate shows the location of the sucrose within the cells.     

DPPH radical scavenging and semicarbazide-sensitive amine oxidase inhibitory and cytotoxic activities of Taiwanofungus camphoratus (Chang-chih)

Wild, liquid state culture and solid state culture of Taiwanofungus camphoratus (Chang-chih) were sequentially extracted with cold water, methanol, and hot water to get cold water soluble, methanol soluble, and hot water soluble extracts respectively. The extracts from three Chang-chih were used to determine 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, semicarbazide sensitive amine oxidase inhibitory, and cytotoxic activities against B16-F10 and HT-1080 cell lines. It was found that extracted fractions from three Chang-chih exhibited the different levels of biological activities.     

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.     

Spinach Chloroplast Membranes after Differential Lipid Extraction

Chloroplasts from spinach were fixed in glutaraldehyde and extracted with three different lipid solvents, after which the lipid composition was analyzed. Studies were also made with the electron microscope. In cold dry acetone, which removes 75 % of lipids, the basic structure of the membranes is unchanged. Acetone with 10 % water removes 89 % of the lipids and a mixture of chloroform with methanol removes 93 % of the lipids, both solvents leaving nearly unrecognizable membrane structures. The relationship between lipid composition and membrane structure is discussed.     

银胶菊叶和花提取物对南方根结线虫的毒杀活性比较

为进一步明确银胶菊(Parthenium hysterophorus L.)的杀线虫活性,对银胶菊叶和花的不同溶剂提取物、甲醇提取物的不同萃取物以及甲醇提取物碱水层的不同极性组分对南方根结线虫(Meloidogyne incognita Chitwood)的杀虫活性进行了检测,并对不同提取物、萃取物和萃取组分进行了生物碱的定性分析.结果表明:银胶菊叶和花的蒸馏水、甲醇、乙酸乙酯和石油醚提取物的得率分别为24.5％和20.3％、19.6％和10.9％、6.8％和7.7％、2.0％和2.7％,其中,叶和花的蒸馏水和甲醇提取物的杀线虫活性均较强,而石油醚提取物的杀线虫活性最弱.用质量体积分数1.0％和0.5％的叶和花蒸馏水提取物分别处理24和48 h后试虫的校正死亡率均达到100.00％;用质量体积分数1.0％和0.5％的叶和花甲醇提取物处理48 h,试虫的校正死亡率均大于90％.叶和花甲醇提取物的碱水层、三氯甲烷Ⅰ层和Ⅱ层萃取物均具有一定的杀线虫活性,其中,用质量体积分数1.0％的花和叶碱水层萃取物以及花的三氯甲烷Ⅰ层萃取物分别处理48 h,试虫的校正死亡率均为100.00％,而三氯甲烷Ⅱ层萃取物的杀线虫活性最弱.银胶菊叶和花甲醇提取物碱水层的11个不同极性组分(A1～A11)也表现出不同程度的杀线虫活性,其中,用质量体积分数0.2％和0.1％花的A2[溶剂为V(三氯甲烷)∶V(甲醇)=10∶1]和A7[溶剂为V(三氯甲烷)∶v甲醇)=1∶1]组分以及叶的A2和A6[溶剂为V(三氯甲烷)∶V(甲醇)=2∶1]组分处理48 h后,试虫的校正死亡率均达100.00％,显著高于其他组分.定性实验结果表明:银胶菊叶和花中具有杀线虫活性的提取物、萃取物和萃取组分中均含有生物碱.研究结果说明:银胶菊花的杀线虫活性高于叶片,其毒杀活性不仅与提取部位及溶剂的种类和极性有关,还与提取物浓度及作用时间等因素有关.     

Floating-Out Technics for Rapid Placement of Ribbons of Serial Sections on Slides

The floating-out technic, popular for single paraffin sections, can be applied successfully to ribbons of serials by either of two procedures. (1) If spreading time for the sections is uncritical suitable lengths of ribbon for attachment to a slide are laid on water at a temperature about 8° C below the melting point of the paraffin and manipulated with a rubber bulb pipette to form a unit. This ensemble can then be picked up by the slide in much the same manner as a single section. (2) If spreading time is critical, as for objects that have had guide limes embedded with them, several ribbons are arranged on a cold, dry slide and transferred to the water as a unit. Placing the ribbons on the cold slide so that they slightly overhang one end and the sides of the slide allows them to make proper contact with the water as the slide is immersed. To facilitate controllable spreading in both methods, the water should have added to it 0.5 ml of albumen-glycerol adhesive per 100 ml. Adding water to the slide after the sections have been picked up or manipulation of the ribbons is generally unnecessary if the ribbons have been aligned accurately on the floating-out bath.     

Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.     

A methanol resorcin-fuchsin stain for elastic tissues and nuclei.

The staining properties of conventional ethanol resorcin-fuchsin and of methanol resorcin-fuchsin were compared. Formula; Dissolve 0.2 g of commercial resorcin-fuschin in 70 ml of methanol or ethanol, add 30 ml of water and 1 m1 of concentrated HC1; stain sections for 4 hours. Both solutions colored elastic and pseudoelastic fibers, cartilage and some mucins. Methanol resorcin-fuchsin also colored nuclei in methacarn- (methanol-chloroform-glacial acetic acid 6:3:1) and formalin-fixed tissues; this nuclear stain withstood counterstaining with picro-dye mictures. Zenker-fixed sections showed diffuse coloration with little or no contrast between nuclei and cytoplasm. Extraction with hot trichloracetic acid abolished binding of methylene blue, but binding of methanol resorcin-fuchsin by nuclei remained unaltered or was enhanced. Experiments with solvents containing various concentrations of methanol, ethanol or isopropanol indicated that the staining patterns of resorcin-fuchsin are determined by the nature and concentration of the alcohol. Methanol resorcin-fuchsin proved useful for simultaneous visualization of elastic tissues and nuclei.     

Methods For Removing Uranyl Acetate Precipitate From Ultrathin Sections

Precipitate resulting from en bloc staining with uranyl acetate was removed by treating sections with 15% oxalic acid in 50% methanol for 30 minutes at 40 C. Precipitate resulting from poststaining sections with hot uranyl acetate was removed by rinsing sections in 0.25-0.50% aqueous oxalic acid for 10-15 seconds at room temperature. Rinsing sections for longer than 30 seconds removed uranyl precipitate and also destained the sections. These procedures did not damage the embedding medium or cellular detail.     

Methods for removing uranyl acetate precipitate from ultrathin sections.

Precipitate resulting from en bloc staining with uranyl acetate was removed by treating sections with 15% oxalic acid in 50% methanol for 30 minutes at 40 C. Precipitate resulting from poststaining sections with hot uranyl acetate was removed by rinsing sections in 0.25-0.50% aqueous oxalic acid for 10-15 seconds at room temperature. Rinsing sections for longer than 30 seconds removed uranyl precipitate and also destained the sections. These procedures did not damage the embedding medium or cellular detail.     

Application of scanning electron microscopy to x-ray analysis of frozen- hydrated sections. I. Specimen handling techniques

X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments. Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions. Use of these techniques should also permit determination of water fraction in cellular compartments. Reliable preparative techniques provide flat, smooth, 0.5 micrometers-thick sections with little elemental and morphological disruption. The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x- ray radiation.     

Effect of Japanese seaweed (Laminaria angustata) extracts on the mutagenicity of 7,12-dimethylbenz[a]anthracene, a breast carcinogen, and of 3,2'-dimethyl-4-aminobiphenyl, a colon and breast carcinogen

Animal model studies suggest that diets containing Laminaria angustata, a brown seaweed commonly eaten in Japan, inhibit breast carcinogenesis. In order to identify the compound(s) in the seaweed responsible for tumor-inhibiting activity, we used Ames/mammalian microsome assay system to determine the antimutagenic (or anticarcinogenic) effect of various solvents and water extracts of Laminaria angustata. The antimutagenic effects of acetone, ether, chloroform, chloroform + methanol, hot water and cold water extracts on the mutagenicity induced by 7,12-dimethylbenz[a]anthracene (DMBA), a breast carcinogen, and 3,2'-dimethyl-4-aminobiphenyl (DMAB), a colon and breast carcinogen, was studied using the Salmonella typhimurium strains TA98 and TA100. All extracts were nonmutagenic in both bacterial tester strains. The addition of 10-100 mg solvent extracts of seaweed/plate greatly inhibited DMAB-induced mutagenicity in both tester strains (80-96% inhibition) and DMBA-induced mutagenicity in TA100 (about 82%), whereas hot and cold water extracts produced a moderate inhibition in a dose-related manner in both strains.