עבודה זו נתמכה על ידי מענקים AG030709 מ-NIH / NIA ו 07-65798 מן המחלקה לבריאות הציבור בקליפורניה. אנו מכירים מרגרט מ Condron לסינתזה פפטיד וניתוח חומצת אמינו, ד&quot;ר אליזבת פ &#39;נויפלד לסיוע ותמיכה בשלבים הראשוניים של הפרויקט, ד&quot;ר צ&#39;י קונג ב חן למתן תמיכה ריאגנטים, וד&quot;ר אנדרו D . אלינגטון לדיונים מועילים.

<ol>
	<li>Monien, B. H., Apostolova, L. G., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+diagnostics+and+therapeutics+for+Alzheimer's+disease-how+early+can+we+get+there.">Early diagnostics and therapeutics for Alzheimer's disease-how early can we get there.</a> Expert. Rev. Neurother. 6, 1293-1306 (2006).</li><li>Nestor, P. J., Scheltens, P., Hodges, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+early+detection+of+Alzheimer's+disease.">Advances in the early detection of Alzheimer's disease.</a> Nat. Med. 10, S34-S41 (2004).</li><li>Kawas, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Early+Alzheimer's+disease.">Clinical practice. Early Alzheimer's disease.</a> N. Engl. J. Med. 349, 1056-1063 (2003).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+&#946;-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid &#946;-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Kirkitadze, M. D., Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigm+shifts+in+Alzheimer's+disease+and+other+neurodegenerative+disorders:+the+emerging+role+of+oligomeric+assemblies.">Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.</a> J. Neurosci. Res. 69, 567-577 (2002).</li><li>Rahimi, F., Shanmugam, A., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+pre-fibrillar+protein+assemblies+in+Alzheimer's+disease+and+related+disorders.">Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders.</a> Curr. Alzheimer Res. 5, 319-341 (2008).</li><li>Jayasena, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+an+emerging+class+of+molecules+that+rival+antibodies+in+diagnostics.">Aptamers: an emerging class of molecules that rival antibodies in diagnostics.</a> Clin. Chem. 45, 1628-1650 (1999).</li><li>Bunka, D. H., Stockley, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers+come+of+age+-+at+last.">Aptamers come of age - at last.</a> Nat. Rev. Microbiol. 4, 588-596 (2006).</li><li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Lee, J. F., Stovall, G. M., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+therapeutics+advance.">Aptamer therapeutics advance.</a> Curr. Opin. Chem. Biol. 10, 282-289 (2006).</li><li>Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+specifically+interact+with+the+prion+protein+PrP.">RNA aptamers specifically interact with the prion protein PrP.</a> J. Virol. 71, 8790-8797 (1997).</li><li>Bibby, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+novel+in+vitro+selection+technique+to+isolate+and+characterise+high+affinity+DNA+aptamers+binding+mammalian+prion+proteins.">Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.</a> J. Virol. Methods. 151, 107-115 (2008).</li><li>Rhie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+2'-fluoro-RNA+aptamers+that+bind+preferentially+to+disease-associated+conformations+of+prion+protein+and+inhibit+conversion.">Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.</a> J. Biol. Chem. 278, 39697-39705 (2003).</li><li>King, D. J., Safar, J. G., Legname, G., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioaptamer+interactions+with+prion+proteins:+sequence-specific+and+non-specific+binding+sites.">Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites.</a> J. Mol. Biol. 369, 1001-1014 (2007).</li><li>Proske, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-protein-specific+aptamer+reduces+PrPSc+formation.">Prion-protein-specific aptamer reduces PrPSc formation.</a> ChemBioChem. 3, 717-725 (2002).</li><li>Murakami, K., Nishikawa, F., Noda, K., Yokoyama, T., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-bovine+prion+protein+RNA+aptamer+containing+tandem+GGA+repeat+interacts+both+with+recombinant+bovine+prion+protein+and+its+&#946;+isoform+with+high+affinity.">Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its &#946; isoform with high affinity.</a> Prion. 2, 73-80 (2008).</li><li>Luhrs, T., Zahn, R., Wuthrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+formation+by+recombinant+full-length+prion+proteins+in+phospholipid+bicelle+solutions.">Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions.</a> J. Mol. Biol. 357, 833-841 (2006).</li><li>Bunka, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+RNA+aptamers+specific+for+amyloid+fibril+epitopes.">Production and characterization of RNA aptamers specific for amyloid fibril epitopes.</a> J. Biol. Chem. 282, 34500-34509 (2007).</li><li>Ylera, F., Lurz, R., Erdmann, V. A., Furste, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+RNA+aptamers+to+the+Alzheimer's+disease+amyloid+peptide.">Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.</a> Biochem. Biophys. Res. Commun. 290, 1583-1588 (2002).</li><li>Rahimi, F., Murakami, K., Summers, J. L., Chen, C. H., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+generated+against+oligomeric+A&#946;40+recognize+common+amyloid+aptatopes+with+low+specificity+but+high+sensitivity.">RNA aptamers generated against oligomeric A&#946;40 recognize common amyloid aptatopes with low specificity but high sensitivity.</a> PLoS ONE. 4, e7694-e7694 (2009).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Rahimi, F., Maiti, P., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+cross-linking+of+unmodified+proteins+(PICUP)+applied+to+amyloidogenic+peptides.">Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.</a> J. Vis. Exp. , (2009).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers-what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers-what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Chen, C. H., Chernis, G. A., Hoang, V. Q., Landgraf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+heregulin+signaling+by+an+aptamer+that+preferentially+binds+to+the+oligomeric+form+of+human+epidermal+growth+factor+receptor-3.">Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3.</a> Proc. Natl. Acad. Sci. USA. 100, 9226-9231 (2003).</li><li>Adams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab math: a handbook of measurements, calculations, and other quantitative skills for use at the bench. , (2003).</li><li>Gopinath, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+developed+for+SELEX.">Methods developed for SELEX.</a> Anal. Bioanal. Chem. 387, 171-182 (2007).</li><li>Takahashi, T., Tada, K., Mihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+selected+against+amyloid+&#946;-peptide+(A&#946;)+inhibit+the+aggregation+of+A&#946;.">RNA aptamers selected against amyloid &#946;-peptide (A&#946;) inhibit the aggregation of A&#946;.</a> Mol. Biosyst. 5, 986-991 (2009).</li></ol>

אין ניגודי אינטרסים הכריז.

נקודת המוצא של התהליך Selex הוא סינתזה של ספריה oligonucleotide אקראי המכיל בדרך כלל 10 -10 12 15 רצפים. ב-DNA Selex, ספריה זו משמשת מיד לאחר בריכה ssDNA שנוצר, ואילו RNA Selex, הפגינו כאן, הספרייה ssDNA מומר הראשון בריכת RNA enzymatically ידי שעתוק חוץ גופית. ואז, Selex מבוצע iteratively לפיה כל מחזור כולל חשיפה המחייב של oligonucleotides ליעד המיועד, חלוקת קלסרים מ בלתי מחייב רצפים, ו elution של רצפי מחייב. בשנת מחזורים מאוחר יותר, stoichiometry יעד ו-RNA ו / או את מספר שוטף ניתן לשנות, או מעכבי תחרותי ניתן להוסיף למאגר מחייב או לשטוף להגדיל את החמרת התנאים Selex. סנן מחייב היא שיטה קלאסי, פשוט, מהיר המשמש Selex 10, אם כי שיטות רבות תוארו 28 עבור מחייב מחיצות. סנן מחייב הוא אידיאלי עבור ללכוד מבחר של RNA-היעד אינטראקציות בתמיסה. כאשר מבחר יעדים הם מולקולות קטנות או פפטידים, גודל הנקבוביות של הממברנה משמש מחייב לסנן הופך להיות הגורם המגביל ואת שיקול חשוב. בעקבות elution, oligonucleotides היעד מחייב הם מוגבר בשימוש מחזורים נוספים. בעת שימוש DNA Selex, הבריכה מועשר יכול להיות מוגבר על ידי ה-PCR ישירות המשמש במחזור הבא לאחר עיכול של רצף ה-DNA משלימים תיוג. בעת שימוש RNA Selex, בריכה זה חייב להיות מומר ל-DNA על ידי שעתוק-הפוך והתרבה ​​PCR, in-vitro עיבד, ומסומן שוב להמשך למחזור הבא. בדרך כלל, 8-20 מחזורים כאלה נדרשים לקבל בריכה aptamer מועשר, אשר לאחר מכן משובטים ו aptamers בודדים רצף ומאופיינת. במחקרים שנעשו באמצעות חלבונים עמילואיד, אפיון aptamers חשוב במיוחד בגלל הזיקה המהותית של oligonucleotides עבור מבנים סיבי עמילואיד פוטנציאל מעכבת התפתחות aptamers ספציפיים שאינם סיבי עמילואיד חלבונים בתנאים פיסיולוגיים 21. זה הטבועה, רצף עצמאית זיקה של oligonucleotides ייתכן הוביל דור של סיבית-cross-reactive aptamers מחקרים במטרה ליצור aptamers שאינם סיבי החלבונים amyloidogenic 17,19-21. לאחרונה, דיווחו טקהאשי et al. דור aptamers RNA נגד מודל oligomeric של Aβ40 והראה תגובתיות עם Aβ monomeric עם זיקה מיקרו 29. עם זאת, תגובתיות צולבת של אלה aptamers עם סיבי Aβ40 או עם חלבונים אחרים סיבי amyloidogenic לא נקבע.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>A&beta;40</td>
<td>&nbsp;</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>Lyophilized powder</td>
</tr>
<tr>
<td>MX5 Automated-S Microbalance</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silicon-coated, 1.6-ml tubes</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19033 or C19035</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Dithiothreitol (DTT)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>D-Salt&trade; Excellulose&trade; desalting columns</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>20449</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate	</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A637-500</td>
<td>&nbsp;</td>
<tr>
<td>Silicon-coated, 0.6-ml tubes</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19063</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10&#x2013;20%)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>10-well; mini size (8 cm X 8 cm); 25 &mu;l loading volume per well; separation range 5 kDa to 40 kDa</td>
</tr>
<tr>
<td>Quartz cuvette</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>105.250-QS</td>
<td>&nbsp;</td>
<tr>
<td>Beckman DU 640 spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ssDNA library</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>The library was designed to contain 49 random nucleotides flanked by two constant regions containing primer-binding and cloning sites: 
5'-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C (N:25:25:25:25%) (N)49 G TCC GTT CGG GAT CCT C-3'</td>
</tr>
<tr>
<td>Taq DNA polymerase</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71160</td>
<td>Recombinant Thermus aquaticus DNA Polymerase supplied with 10&#215; PCR Buffer and a separate tube of 25 mM MgCl2 for routine PCR.</td>
</tr>
<tr>
<td>PCR Nucleotide Mix, 10 mM solution</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>77212</td>
<td>(10 mM each dATP, dCTP, dGTP, dTTP)</td>
</tr>
<tr>
<td>Forward primer</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>5'-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C-3'</td>
</tr>
<tr>
<td>Reverse primer</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>5'-GAG GAT CCC GAA CGG AC-3'</td>
</tr>
<tr>
<td>Thermal cycler</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>Techne TC-312</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit (50)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>CA3510-8</td>
<td>&nbsp;</td>
<tr>
<td>Conical, sterile 1.6-ml tubes with caps attached with O-rings</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19040-S</td>
<td>&nbsp;</td>
<tr>
<td>RiboMAX&trade; Large Scale RNA Production System&#x2013;T7</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>P1300</td>
<td>The kit contains: 120 &mu;l Enzyme Mix (RNA polymerase, recombinant RNasin&reg; ribonuclease inhibitor and recombinant inorganic pyrophosphatase); 240 &mu;l transcription 5 buffer; 100 &mu;l each of 4 rNTPs, 100 mM; 110 U RQ1 RNase-free DNase, 1 U/&mu;l; 10 &mu;l linear control DNA, 1 mg/ml; 1 ml 3M sodium acetate (pH 5.2); 1.25 ml nuclease-Free water</td>
</tr>
<tr>
<td>&alpha;-32P-cytidine 5'-triphosphate, 250 &mu;Ci (9.25 MBq), </td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>BLU008H250UC</td>
<td>Specific Activity: 3000 Ci (111 TBq)/mmol, 50 mM Tricine (pH 7.6)</td>
</tr>
<tr>
<td>Citrate-saturated phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.7)</td>
<td>&nbsp;</td>
<td>Sigma (Fluka)</td>
<td>77619</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform:Isoamyl alcohol (24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0549</td>
<td>&nbsp;</td>
<tr>
<td>Absolute ethanol for molecular biology</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>&nbsp;</td>
<tr>
<td>Z216-MK refrigerated microcentrifuge</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C0216-MK</td>
<td>&nbsp;</td>
<tr>
<td>illustra ProbeQuant&trade; G-50 Micro Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>Obtained from Fisher Scientific (45-001-487)</td>
<td>Prepacked with Sephadex&trade; G-50 DNA Grade and pre-equilibrated in STE buffer containing 0.15% Kathon as Biocide</td>
</tr>
<tr>
<td>Triathler Bench-top Scintillation counter</td>
<td>&nbsp;</td>
<td>Hidex Oy, Turku, Finland</td>
<td>Triathler LSC Model: 425-034</td>
<td>&nbsp;</td>
<tr>
<td>Novex&reg; TBE-Urea Sample Buffer (2&#215;)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6876</td>
<td>&nbsp;</td>
<tr>
<td>6% TBE-Urea Gels 1.0 mm, 10 wells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6865BOX</td>
<td>&nbsp;</td>
<tr>
<td>Novex&reg; TBE Running Buffer (5&#215;)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6675</td>
<td>&nbsp;</td>
<tr>
<td>Radioactivity decontaminant</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>04-355-67</td>
<td>&nbsp;</td>
<tr>
<td>Gel-loading tips</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>P3080</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>XCell SureLock Mini-Cell</td>
</tr>
<tr>
<td>Autoradiography film</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>E3018</td>
<td>Use in complete darkness</td>
</tr>
<tr>
<td>Autoradiography film, Hyperfilm&trade; ECL</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>RPN3114K</td>
<td>Can be used under red safe light.</td>
</tr>
<tr>
<td>Membrane discs</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>GSWP02500</td>
<td>Mixed cellulose ester, hydrophilic, 0.22-&mu;m disc membranes</td>
</tr>
<tr>
<td>Fritted glass support base for 125-ml flask</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>26316-696</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-11YZ</td>
<td>&nbsp;</td>
<tr>
<td>Urea	</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC32738-0050</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>118430010</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1767</td>
<td>&nbsp;</td>
<tr>
<td>2-Propanol for molecular biology</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I9516</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant RNase inhibitor</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71571</td>
<td>&nbsp;</td>
<tr>
<td>ImProm-II&trade;Reverse Transcription System</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A3802</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant RNase inhibitor</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71571</td>
<td>&nbsp;</td>
<tr>
<td>RapidRun&trade; Loading Dye</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>77524</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

selection of aptamers for amyloid &beta;-protein, the causative agent of alzheimer&#39;s disease

מחלת אלצהיימר (AD) הוא פרוגרסיבי, תלויי גיל, הפרעת ניווניות עם קורס חתרני שהופכת presymptomatic האבחנה שלה קשה 1. אבחנה ברורה לספירה מושגת רק נתיחה שלאחר המוות, ובכך לבסס אבחנה presymptomatic, מוקדם של אלצהיימר הוא חיוני לפיתוח טיפולים יעילים לניהול 2,3. עמילואיד β-חלבון (Aβ) הוא מרכזי בהיווצרות לספירה. מסיסים, מכלולים Aβ oligomeric הם האמינו הבסיסית neurotoxicity להשפיע על תפקוד סינפטי ואיבוד נוירון לספירה 4,5. צורות שונות של מכלולים Aβ מסיסים תוארו, לעומת זאת, יחסי הגומלין שלהם רלוונטיות האטיולוגיה לספירה בפתוגנזה הם מורכבים ולא הבין היטב 6. כלים ספציפיים ההכרה המולקולרית עשויה לפתור את היחסים בין מכלולים Aβ ולאפשר זיהוי ואפיון של מכלולים אלה מוקדם במהלך המחלה לפני שהתסמינים מופיעים. הכרה מולקולרית בדרך כלל מסתמכת על נוגדנים. עם זאת, מעמד חלופי של כלי הכרה, aptamers מולקולרית, מציעה יתרונות חשובים ביחס נוגדנים 7,8. Aptamers הם oligonucleotides שנוצר על ידי בחירה ב-vitro: התפתחות שיטתית של ligands על ידי העשרה מעריכי (Selex) 9,10. Selex היא תהליך חוזר ונשנה זה, בדומה לאבולוציה הדרווינית, מאפשר בחירה, הגברה, העשרה, והנצחה של הנכס, למשל, נלהב, ספציפית, מחייב ליגנד (aptamers) או פעילות קטליטית (ribozymes ו DNAzymes). למרות הופעתה של aptamers ככלים בתחום הביוטכנולוגיה והרפואה המודרנית 11, הם כבר לא מנוצלים בתחום עמילואיד. RNA מעטים או aptamers ssDNA נבחרו נגד צורות שונות של חלבונים Prion (PRP) 12-16. Aptamer RNA שנוצר נגד PRP רקומביננטי שור הוצגה להכיר שור PRP-β 17, מסיסים, oligomeric, גיליון β-עשירים גרסה קונפורמציה של PRP באורך מלא שנוצר סיבי עמילואיד 18. Aptamers שנוצר באמצעות טפסים monomeric וכמה סיבי β 2-microglobulin (β 2 מ &#39;) נמצאו לאגד הסיבים של חלבונים מסוימים amyloidogenic אחרים מלבד β 2 מ&#39; הסיבים 19. Ylera et al. תיאר aptamers RNA שנבחר נגד משותקת monomeric Aβ40 20. באופן בלתי צפוי, aptamers אלה קשורים סיבי Aβ40. בסך הכל, הנתונים הללו מעלים שאלות חשובות. מדוע נבחר aptamers נגד חלבונים monomeric לזהות צורות פולימריות שלהם? יכול aptamers נגד צורות monomeric ו / או oligomeric של חלבונים amyloidogenic לקבל? כדי לענות על השאלות האלה, ניסינו לבחור aptamers עבור קוולנטית מיוצב oligomeric Aβ40 21 שנוצר באמצעות תמונה-Induced cross-linking של חלבונים ללא שינוי (PICUP) 22,23. בדומה לממצאים קודמים 17,19,20, aptamers אלה הגיבו הסיבים של Aβ וכמה חלבונים אחרים amyloidogenic סביר הכרה עמילואיד משותף פוטנציאלי מבניים aptatope 21. כאן, אנו מציגים את המתודולוגיה Selex המשמשים לייצור של אלה aptamers 21.

Watch this Scientific Journal Video about Selection of Aptamers for Amyloid Î²-Protein, the Causative Agent of Alzheimer's Disease at JoVE.com

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers הם ribo-/deoxyribo-oligonucleotides קצר נבחר על ידי<em&gt; חוץ גופית</em&gt; שיטות האבולוציה מבוסס על זיקה יעד ספציפי. Aptamers הם כלים לזיהוי מולקולרי עם יישומים טיפולית, אבחון, מחקר מגוונות. אנו מדגימים שיטות הבחירה של aptamers לחלבון β עמילואיד, הסוכן סיבתי של מחלת אלצהיימר.

מבחר Aptamers עבור חלבון β עמילואיד, הסוכן סיבתי של מחלת אלצהיימר

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

19.3K Views.  David Geffen School of Medicine. Aptamers הם ribo-/deoxyribo-oligonucleotides קצר נבחר על ידי חוץ גופית שיטות האבולוציה מבוסס על זיקה יעד ספציפי. Aptamers הם כלים לזיהוי מולקולרי עם יישומים טיפולית, אבחון, מחקר מגוונות. אנו מדגימים שיטות הבחירה של aptamers לחלבון β עמילואיד, הסוכן סיבתי של מחלת אלצהיימר.

Video: Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

איתור של הפלאק מדלקת עצבים במודל מחלת אלצהיימר עכבר

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

זיהוי של היפוקסיה Microregional בקליפת עכבר מוחין על ידי הדמיה של שני הפוטונים אנדוגני NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

הר immunolabeling השלם של חוש הריח קולטן נוירונים<em&gt; דרוזופילה</em&gt; אנטנה

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

מבחני חוש הריח למודלי עכבר של ניווניות מחלות

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

עירוי stereotaxic של Oligomeric עמילואיד-ביתא להיפוקמפוס העכבר

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

למה כימות Matters: אפיון פנוטיפים בבית<em&gt; תסיסנית</em&gt; זחל Neuromuscular צומת

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

אין ויוו מעקב אחר בתחנת האוטובוסים Presynaptic עצבים מוטוריים דרוזופילה מולקולה בודדת

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

ויזואליזציה של הפקדות β עמילואיד במוח האנושי עם Desorption לייזר בסיוע מטריקס/הדמיה ספקטרומטר מסה יינון

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

תיוג והדמיה של שלטי עמילואיד ברקמת המוח באמצעות פוליפנול טבעי כורכומין

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

הערכה סטריאוולוגית של אורך סיבים Cholinergic בגרעין באסליס של מיינרט של המוח עכבר

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...

מבחר Aptamers עבור חלבון β עמילואיד, הסוכן סיבתי של מחלת אלצהיימר

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