scholarly journals Effects of Detergents on the Redistribution of Gangliosides and GPI-anchored Proteins in Brain Tissue Sections

2007 ◽  
Vol 55 (8) ◽  
pp. 805-812 ◽  
Author(s):  
Marija Heffer-Lauc ◽  
Barbara Viljetić ◽  
Katarina Vajn ◽  
Ronald L. Schnaar ◽  
Gordan Lauc
Keyword(s):  
Cell Membrane ◽  
Brain Tissue ◽  
Room Temperature ◽  
Deoxycholic Acid ◽  
Tween 20 ◽  
Outer Side ◽  
Tissue Sections ◽  
Brij 96V ◽  
Triton X ◽  
The Ideal

Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of another cell. Recently, we reported that under conditions of lipid raft isolation, Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent, we evaluated the effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on brain tissue sections. At room temperature, all detergents (1% concentration) extracted significant amounts of both gangliosides and Thy-1. At 4C, the extraction was weaker, but Triton X-100, CHAPS, and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from gray matter into the white matter. Both redistribution and extraction were significantly augmented when sections were incubated with detergents in the presence of primary antibodies. Of the nine tested detergents, none is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes while causing the least amount of artifacts. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials. (J Histochem Cytochem 55: 805–812, 2007)

Immumohistochemical Detection and Localization of Prion Protein in Brain Tissue of Sheep With Natural Scrapie

1995 ◽  
Vol 32 (3) ◽  
pp. 299-308 ◽  
Author(s):  
L. J. M. van Keulen ◽  
B. E. C. Schreuder ◽  
R. H. Meloen ◽  
M. Poelen-van den Berg ◽  
G. Mooij-Harkes ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Brain Tissue ◽  
Prion Protein ◽  
Medulla Oblongata ◽  
Brain Regions ◽  
Cellular Prion Protein ◽  
Immunohistochemical Method ◽  
Tissue Sections ◽  
Natural Scrapie ◽  
Detection And Localization

A converted form of the normal cellular prion protein (PrP) accumulates in the brains of sheep with scrapie. We describe an immunohistochemical method for identifying scrapie-associated PrP (PrPSc) in periodate-lysine-paraformaldehyde-fixed brain tissue, which provides adequate preservation of tissue morphology. After pretreatment of tissue sections with formic acid and hydrated autoclaving, we located PrPSc in the brains of 50 sheep with natural scrapie by use of antipeptide antisera raised against ovine PrP. No PrP was seen in 20 sheep without histopathologic signs of scrapie. PrP80 that did not stain for amyloid was present in the cytoplasm and at the cell membrane of both neurons and astrocytes. Large amounts of PrPSc were seen at the cell membrane of neurons in the medulla oblongata and pons, whereas PrPSc accumulated at the cell membrane of astrocytes of the glial limitans in all brain regions. PrPSc that stained for amyloid was located in the walls of blood vessels and perivascularly in the brains of 32 (64%) of 50 sheep, mainly in the thalamus and never in the pons or medulla oblongata. No apparent topographic relationship existed between PrPSc that stained for amyloid and PrPSc accumulation associated with neurons or astrocytes. In all scrapie-affected sheep, PrPSc was present in brain regions with vacuolation, but it could also be detected in regions with minimal or no vacuolation. We conclude that the immunohistochemical detection of PrP can be an important confirmative test in scrapie diagnosis.

Lobular and cellular distribution and content of glycogen synthase in periportal and pericentral hepatocytes of the rat liver

1993 ◽  
Vol 51 ◽  
pp. 394-395
Author(s):  
Bruce F. Giffin ◽  
Randal E. Morris ◽  
Richard L. Drake ◽  
Robert R. Cardell
Keyword(s):  
Rat Liver ◽  
Room Temperature ◽  
Fetal Calf Serum ◽  
Glycogen Synthase ◽  
Calf Serum ◽  
Cellular Distribution ◽  
Heterogeneous Distribution ◽  
Tissue Sections ◽  
Rabbit Igg ◽  
Triton X

Glycogen and the enzymes involved in hepatic carbohydrate metabolism have a heterogeneous distribution throughout the parenchyma of the liver. Although the precise lobular localization for many of these enzymes has been determined, information on the distribution and content of glycogen synthase (GS) has not been published. These studies were undertaken to determine: (1) the lobular and cellular distribution of GS in the fed rat; (2) any relationship between glycogen patterns and the distribution of GS.Livers of fed male Spraque-Dawley rats were perfused with cold 30% sucrose and 10 um frozen sections collected on Vectabond treated slides. After fixation for 5 min in 4% paraformaldehyde at room temperature, tissue sections were incubated in polyethylene slide boxes with blocking buffer (PBS, 0.5% Triton X-100, 5% fetal calf serum, 2% bovine serum albumin, 0.95% fish gelatin, pH 7.5) overnight at 4°C. Tissue sections were incubated for 6 hours at room temperature with goat anti-rat liver GS diluted in blocking buffer (1:1000) followed by 5 nm gold conjugated rabbit anti-goat IgG overnight at 4°C and 3 hours with 5 nm gold conjugated goat anti-rabbit IgG at room temperature.

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

1987 ◽  
Vol 65 (1) ◽  
pp. 8-18 ◽  
Author(s):  
Rex K. M. Wong ◽  
Christine P. Nichol ◽  
M. Chandra Sekar ◽  
Basil D. Roufogalis
Keyword(s):  
Chain Length ◽  
Membrane Lipids ◽  
Acetylcholinesterase Activity ◽  
Exchange Chromatography ◽  
Erythrocyte Membranes ◽  
Tween 20 ◽  
Bovine Erythrocyte ◽  
Critical Micelle Concentrations ◽  
Triton X ◽  
Nonionic Detergents

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octy1 β-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70–80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

Visualisation of GABAergic neurons and synapses in the rat brain using immunohistochemistry for two forms of glutamate decarboxylase

2021 ◽  
Vol 21 (2) ◽  
pp. 63-73
Author(s):  
Valeria A. Razenkova ◽  
Dmitrii E. Korzhevskii
Keyword(s):  
Brain Tissue ◽  
Glutamate Decarboxylase ◽  
Brain Structures ◽  
Gabaergic Neurons ◽  
Laboratory Animals ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Background Staining ◽  
The Central Nervous System ◽  
Rat Forebrain

BACKGROUND: Taking into account the importance of GABAergic brain system research and also the opportunity to achieve specific and accurate results in laboratory studies using immunohistochemical approaches, it seems important to have a reliable method of visualization GABA-synthesizing cells, their projections and synapses, for the morphofunctional analysis of GABAergic system both in normal conditions and in the experimental pathology. AIM: The aim of the study was to visualize analyze GABAergic neurons and synapses within rats brain using three different antibody types against glutamate decarboxylase and to identify the optimal conditions for reaction performing. MATERIALS AND METHODS: The study was performed on paraffin brain tissue sections of 5 adult Wistar rats. Immunohistochemical reactions using three antibody types against glutamate decarboxylase isoform 67 (GAD67) and glutamate decarboxylase isoform 65 (GAD65) were performed. Additional controls on C57/Bl6 mice and Chinchilla rabbits brain samples were also carried out. RESULTS: Antibodies used in the research made it possible to achieve high quality of GABAergic structures visualizing without increasing background staining. At the same time different antibody types are distinct in their efficacy to perform immunohistochemistry reaction on laboratory animal brain tissue samples. By performing additional controls, we discovered that there is necessary to adsorb secondary reagents immunoglobulins in order to eliminate nonspecific staining. It was found that GAD67 and GAD65 distribution in rat forebrain structures is different. It was stated that GAD67 immunohistochemistry most completely reveals GABAergic brain structures compared to GAD65 immunhistochemistry. The possibility of determining morphological features of GABAergic neurons and synaptic terminals, as well as performing quantitative analysis, was demonstrated. CONCLUSIONS: The approach proposed makes it possible to specifically visualize GABAergic structures of the central nervous system of different laboratory animals. This could be useful both in fundamental studies and in pathology research.

Effect of Hot Processing on Mircrostructure and Properties of Flat Billets of TA15 Titanium Alloy

2021 ◽  
Vol 1016 ◽  
pp. 906-910
Author(s):  
Xin Hua Min ◽  
Cheng Jin
Keyword(s):  
Mechanical Properties ◽  
Heat Treatment ◽  
Titanium Alloy ◽  
High Temperature ◽  
Room Temperature ◽  
High Strength ◽  
Slow Cooling ◽  
Microstructure And Mechanical Properties ◽  
Ta15 Titanium Alloy ◽  
The Ideal

In this paper,effect of the different forging processes on the microstructure and mechanical properties of the flat flat billets of TA15 titanium alloy was investigated.The flat billiets of 80 mm×150 mm×L sizes of TA15 titanium alloy are produced by four different forging processes.Then the different microstrure and properties of the flat billiets were obtained by heat treatment of 800 °C~850 °C×1 h~4h.The results show that, adopting the first forging temperature at T1 °C、slow cooling and the second forging temperature at T2°C 、quick cooling, the primary αphases content is just 10%, and there are lots of thin aciculate phases on the base. This microstructure has both high strength at room temperature and high temperature, while the properties between the cross and lengthwise directions are just the same. So the hot processing of the first forging temperature at T1 °C、slow cooling and the second forging temperature at T2°C 、quick cooling is choosed as the ideal processing for production of aircraft frame parts.

Improvement on the Use of 3-aminopropyltriethoxysilane and Poly-L-lysine Coated Slides for Immunohistochemical Staining of Lifting Tissue Sections

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S114-S114
Author(s):  
S Iyiola ◽  
O Aremu ◽  
J A Onifade
Keyword(s):  
Brain Tissue ◽  
Tissue Section ◽  
University Teaching ◽  
Antigen Retrieval ◽  
Buffer Solution ◽  
Citrate Buffer ◽  
Tissue Sections ◽  
The Brain ◽  
Control Post ◽  
Control Study

Abstract Introduction/Objective Lifting of tissue section during antigen retrieval occasionally occur most especially with the brain tissue. therefore, this work seeks to provide solution on how to use locally charged 3-aminopropyltriethoxysilane (APES) and poly-l-lysine coated slides for brain immunohistochemistry (IHC). Methods This laboratory experimental control study was carried out in Obafemi Awolowo University Teaching Hospital Complex, Nigeria owing to section lifting associated with our brain tissue during IHC. Two tissue blocks of lifting brain section were cut at 5um. 30 sections were cut from each block. 10 sections from each block were floated onto APES coated slides, poly-l-lysine and superfrost plus. The sections were allowed to stand for two hours before putting them on hot plate at 370C overnight. The slides were arranged on 4 slide carrier containing 15 slides from each block, 5 slides from each group. The slides were dewaxed and hydrated to distilled water. Then, a set of 15 slides in a carrier from each block was allowed to air dry under ceiling fan on hot plates at 30OC until no traces of water was found on them. All the slides were then subjected to heat mediated antigen retrieval protocol using citrate buffer solution. Three slides from each block consisting of a non-post hydration air dried tissue section on superfrost plus as a control, post hydration air dried tissue section from APES and poly-l-lysine were stained with GFAP. Results All the sections on superfrost plus did not lift. All the post hydration air dried tissue sections on locally charged slides did not lift. Approximately one tissue section from non-air dried post hydration locally charged slides did not lift. There was no difference in the staining pattern of GFAP on all the tissue sections. Conclusion We propose that air dried tissue section after dewax and hydration could be used for GFAP study, most especially in a population that cannot afford commercially charged slides.

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