Support for this study came from the Field Neurosciences Institute at Ascension of St. Mary&#39;s.

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Saginaw Valley State University. The human tissue was obtained from an established brain bank at the Banner Sun Health Institute in Arizona15,16.
1. Perfusion of the animals
<ol>
	<li>Prepare the fixative and perfusion buffers.
	<ol>
		<li>Prepare 0.1 M sodium phosphate buffer by adding 80 g of sodium chloride (NaCl), 2 g of potassium chlorid...

<ol>
	<li>Cummings, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> New England Journal of Medicine. 351 (1), 56-67 (2004).</li><li>Jack, C. R., Holtzman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarker+modeling+of+Alzheimer's+disease.">Biomarker modeling of Alzheimer's disease.</a> Neuron. 80 (6), 1347-1358 (2013).</li><li>Tarawneh, R., Holtzman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+problem+of+symptomatic+Alzheimer+disease+and+mild+cognitive+impairment.">The clinical problem of symptomatic Alzheimer disease and mild cognitive impairment.</a> Cold Spring Harbor Perspectives in Medicine. 2 (5), (2012).</li><li>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=Cell+biology+of+protein+misfolding:+the+examples+of+Alzheimer's+and+Parkinson's+diseases.">Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases.</a> Nature Cell Biology. 6 (11), 1054-1061 (2004).</li><li>Hardy, J., Allsop, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+deposition+as+the+central+event+in+the+aetiology+of+Alzheimer's+disease.">Amyloid deposition as the central event in the aetiology of Alzheimer's disease.</a> Trends in Pharmacological Sciences. 12 (10), 383-388 (1991).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+curcumin+in+diagnosis,+prevention,+and+treatment+of+Alzheimer's+disease.">Use of curcumin in diagnosis, prevention, and treatment of Alzheimer's disease.</a> Neural Regeneration Research. 13 (4), 742-752 (2018).</li><li>Maiti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+dietary+curcumin,+nanocurcumin,+and+other+classical+amyloid-binding+dyes+for+labeling+and+imaging+of+amyloid+plaques+in+brain+tissue+of+5x-familial+Alzheimer's+disease+mice.">A comparative study of dietary curcumin, nanocurcumin, and other classical amyloid-binding dyes for labeling and imaging of amyloid plaques in brain tissue of 5x-familial Alzheimer's disease mice.</a> Histochemistry and Cell Biology. 146 (5), 609-625 (2016).</li><li>Maiti, P., Dunbar, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Curcumin,+a+Natural+Polyphenol+for+Targeting+Molecular+Pathways+in+Treating+Age-Related+Neurodegenerative+Diseases.">Use of Curcumin, a Natural Polyphenol for Targeting Molecular Pathways in Treating Age-Related Neurodegenerative Diseases.</a> International Journal of Molecular Sciences. 19 (6), (2017).</li><li>Maiti, P., Dunbar, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Neuroprotective+Effects+of+Dietary+Curcumin+and+Solid+Lipid+Curcumin+Particles+in+Cultured+Mouse+Neuroblastoma+Cells+after+Exposure+to+Abeta42.">Comparative Neuroprotective Effects of Dietary Curcumin and Solid Lipid Curcumin Particles in Cultured Mouse Neuroblastoma Cells after Exposure to Abeta42.</a> International Journal of Alzheimer's Disease. , (2017).</li><li>den Haan, J., Morrema, T. H. J., Rozemuller, A. J., Bouwman, F. H., Hoozemans, J. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+curcumin+forms+selectively+bind+fibrillar+amyloid+beta+in+post+mortem+Alzheimer's+disease+brains:+Implications+for+in-vivo+diagnostics.">Different curcumin forms selectively bind fibrillar amyloid beta in post mortem Alzheimer's disease brains: Implications for in-vivo diagnostics.</a> Acta Neuropathologica Communications. 6 (1), 75 (2018).</li><li>Koronyo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+amyloid+pathology+and+proof-of-concept+imaging+trial+in+Alzheimer's+disease.">Retinal amyloid pathology and proof-of-concept imaging trial in Alzheimer's disease.</a> JCI Insight. 2 (16), (2017).</li><li>Koronyo, Y., Salumbides, B. C., Black, K. L., Koronyo-Hamaoui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+in+the+retina:+imaging+retinal+abeta+plaques+for+early+diagnosis+and+therapy+assessment.">Alzheimer's disease in the retina: imaging retinal abeta plaques for early diagnosis and therapy assessment.</a> Neurodegenerative Diseases. 10 (1-4), 285-293 (2012).</li><li>Koronyo-Hamaoui, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+amyloid+plaques+in+retinas+from+Alzheimer's+patients+and+noninvasive+in+vivo+optical+imaging+of+retinal+plaques+in+a+mouse+model.">Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model.</a> NeuroImage. 54 (Suppl 1), S204-S217 (2011).</li><li>Ran, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+synthesis,+and+testing+of+difluoroboron-derivatized+curcumins+as+near-infrared+probes+for+in+vivo+detection+of+amyloid-beta+deposits.">Design, synthesis, and testing of difluoroboron-derivatized curcumins as near-infrared probes for in vivo detection of amyloid-beta deposits.</a> Journal of the American Chemical Society. 131 (42), 15257-15261 (2009).</li><li>Beach, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sun+Health+Research+Institute+Brain+Donation+Program:+Description+and+Experience,+1987-2007.">The Sun Health Research Institute Brain Donation Program: Description and Experience, 1987-2007.</a> Cell Tissue Bank. 9 (3), 229-245 (2008).</li><li>Green, S. J., Killiany, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subregions+of+the+inferior+parietal+lobule+are+affected+in+the+progression+to+AD.">Subregions of the inferior parietal lobule are affected in the progression to AD.</a> Neurobiology of Aging. 31 (8), 1304-1311 (2010).</li><li>Ono, K., Hasegawa, K., Naiki, H., Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+has+potent+anti-amyloidogenic+effects+for+Alzheimer's+beta-amyloid+fibrils+in+vitro.">Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.</a> Journal of Neuroscience Research. 75 (6), 742-750 (2004).</li><li>Garcia-Alloza, M., Borrelli, L. A., Rozkalne, A., Hyman, B. T., Bacskai, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+labels+amyloid+pathology+in+vivo,+disrupts+existing+plaques,+and+partially+restores+distorted+neurites+in+an+Alzheimer+mouse+model.">Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model.</a> Journal of Neurochemistry. 102 (4), 1095-1104 (2007).</li><li>Mutsuga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+curcumin+to+senile+plaques+and+cerebral+amyloid+angiopathy+in+the+aged+brain+of+various+animals+and+to+neurofibrillary+tangles+in+Alzheimer's+brain.">Binding of curcumin to senile plaques and cerebral amyloid angiopathy in the aged brain of various animals and to neurofibrillary tangles in Alzheimer's brain.</a> Journal of Veterinary Medical Science. 74 (1), 51-57 (2012).</li><li>Tei, M., Uchida, K., Mutsuga, M., Chambers, J. K., Nakayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+of+curcumin+to+various+types+of+canine+amyloid+proteins.">The binding of curcumin to various types of canine amyloid proteins.</a> Journal of Veterinary Medical Science. 74 (4), 481-483 (2012).</li><li>Liu, L., Komatsu, H., Murray, I. V., Axelsen, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+amyloid+beta+protein+misfolding+and+fibrillogenesis+by+a+lipid+oxidation+product.">Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product.</a> Journal of Molecular Biology. 377 (4), 1236-1250 (2008).</li><li>Wu, C., Scott, J., Shea, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+Congo+red+to+amyloid+protofibrils+of+the+Alzheimer+Abeta(9-40)+peptide+probed+by+molecular+dynamics+simulations.">Binding of Congo red to amyloid protofibrils of the Alzheimer Abeta(9-40) peptide probed by molecular dynamics simulations.</a> Biophysical Journal. 103 (3), 550-557 (2012).</li><li>Wu, C., Wang, Z., Lei, H., Zhang, W., Duan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+binding+modes+of+Congo+red+to+amyloid+protofibril+surface+observed+in+molecular+dynamics+simulations.">Dual binding modes of Congo red to amyloid protofibril surface observed in molecular dynamics simulations.</a> Journal of the American Chemical Society. 129 (5), 1225-1232 (2007).</li><li>Gutierrez, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+Method+to+Detect+Neuronal+Degeneration+and+Amyloid+beta+Accumulation+in+Free-Floating+Brain+Sections+With+Fluoro-Jade.">Alternative Method to Detect Neuronal Degeneration and Amyloid beta Accumulation in Free-Floating Brain Sections With Fluoro-Jade.</a> ASN Neuro Methods. 10, 1-7 (2018).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+inhibits+formation+of+amyloid+beta+oligomers+and+fibrils,+binds+plaques,+and+reduces+amyloid+in+vivo.">Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo.</a> Journal of Biological Chemistry. 280 (7), 5892-5901 (2005).</li></ol>

Our hypothesis was that Cur could be used as the quickest, easiest, and least expensive way to label and image A&#946; plaques in postmortem AD brain tissue when compared to other classical amyloid binding dyes, as well as A&#946;-specific antibodies. The aims of this study were to determine the minimum time required to label and image A&#946; plaques by Cur in postmortem AD brain tissue and determine whether Cur can be used as an alternative to A&#946; antibody for labeling A&#946; plaques. To this end, the A&#946;-labe...

Alzheimer&#39;s disease (AD) is one of the most common, age-related, progressive neurological disorders and one of the leading causes of death worldwide1,2. Learning, memory, and cognition impairment, along with neuropsychiatric disorders, are the common symptoms manifested in AD3. Although the etiology of AD has not been fully elucidated, the available genetic, biochemical, and experimental evidence indicates that gradual deposition of A&#946; is a definitive biomarker for AD4. This misfolded protein accumulates in intracellular and extracellular spaces and is thought to be involved in synaptic loss, increased neuroinflammation, and neurodegeneration in the cortical and hippocampal regions in brain affected by AD5. Therefore, histochemical detection of A&#946; in AD tissue is a crucial first step in developing non-toxic, anti-amyloid drugs to prevent AD progression.
During the past few decades, several dyes and antibodies have been used by many research laboratories to label and image A&#946; plaques in brain tissue, but some of these methods are time consuming and the dyes or antibodies used are expensive, requiring several accessory chemicals. Therefore, the development of an inexpensive means of detection of A&#946; plaques in the AD brain would be a welcome new tool. Many laboratories started using Cur, a promising anti-amyloid natural polyphenol, for labeling and imaging A&#946;, as well as a therapeutic agent for AD6,7,8,9. Its hydrophobicity and lypophilic nature, structural similarities with classical amyloid binding dyes, strong fluorescent activity, as well as strong affinity to bind with A&#946; makes it an ideal fluorophore for labeling and imaging of A&#946; plaques in AD tissue10. Cur binds with A&#946;-plaques and oligomers and its presence is also detected in intracellular spaces7,11,12,13. In addition, it has been shown that minimal amounts (1&#8722;10 nM) of Cur can label A&#946; plaques in 5x familial Alzheimer&#39;s disease (5xFAD) brain tissue7. Although the 1 nM concentration does not provide the optimal fluorescence intensity for counting of A&#946; plaques, a 10 nM or higher concentration of Cur does. Ran and colleagues14 reported that doses as low as 0.2 nM of difluoroboron-derivatized Cur can detect in vivo A&#946; deposits nearly as well as an infrared probe. Whether this dose is sufficient to label A&#946; plaques in tissue is still not clear. Most previous studies have used 20&#8722;30 min for staining A&#946; plaques using Cur, but optimal staining may require much less time.
The present study was designed to test the minimum time required by Cur to label A&#946; plaques in AD brain tissue and to compare the sensitivity for labeling and imaging of A&#946; plaques in brain tissue from the 5xFAD mice after staining with Cur with other conventional A&#946;-binding dyes, such as Thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC). The A&#946; labeling capability of these classical amyloid binding dyes was compared with Cur staining in paraffin-embedded and cryostat coronal brain sections from 5xFAD mice and from aged-matched human AD and control brain tissue. The findings suggest that Cur labels A&#946; plaques in a manner similar to A&#946;-specific antibodies (6E10) and moderately better than Thio-S, CR, or FJC. In addition, when intraperitoneal injections of Cur to 5xFAD mice were administered for 2&#8722;5 days, it crossed the blood-brain barrier and bound with A&#946; plaques7. Interestingly, nanomolar concentrations of Cur have been used to label and image A&#946; plaques in 5xFAD brain tissue7,14. Moreover, morphologically distinct A&#946; plaques, such as core, neuritic, diffuse, and burned-out plaques can be labeled by Cur more efficiently than with any of the other conventional amyloid binding dyes7. Overall, Cur can be applied to label and image A&#946; plaques in postmortem brain tissue from AD animal models and/or human AD tissue in an easy and inexpensive way, as a reliable alternative to A&#946;-specific antibodies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>IHC world, Woodstock, MD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aanimal model of Alzheimer&#39;s disease</td>
			<td>Jackson&#39;s laboratory, Bar Harbor, ME</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute alcohol</td>
			<td>VWR,Radnor, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 594</td>
			<td>Santacruz Biotech, Dallas, TX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody 6E10</td>
			<td>Biolegend, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody A11</td>
			<td>Millipore, Burlington, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound light microscope</td>
			<td>Olympus, Shinjuku, Japan</td>
			<td>Olympus BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Congo red</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>GMI, Ramsey, MN</td>
			<td>LeicaCM1800</td>
			<td></td>
		</tr>
		<tr>
			<td>Curcumin</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dystyrene plasticizer xylene</td>
			<td>BDH, Dawsonville, GA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter papers</td>
			<td>Fisher scientific, Pittsburgh, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Leica, Buffalo Grove, IL</td>
			<td>Leica DMI 6000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus, Shinjuku, Japan</td>
			<td>Olympus 1x70</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ploy-lysine coated charged glass slide</td>
			<td>Globe Scientific Inc, Mahwah, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>EMD Millipore, Burlington, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Vortex Pharmaceuticals limited, Dearborn, MI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin-S</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma, St. Louis, MO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR,Radnor, PA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 labels A&#946; plaques within a minute. When we stained 5xFAD tissue with Cur, we found that Cur label A&#946; plaques within 1 min. Although increased incubation time with Cur slightly increased the fluorescence intensity of A&#946; plaques, the number of observed A&#946; plaques was not significantly different between 1 min and 5 min staining time (Figure 1).
Cur can label A&#946; plaques...

Watch this Scientific Journal Video about Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin at JoVE.com

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

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.

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

This protocol is the most efficient and cost-effective method for accurately labeling beta-amyloid plaques, as compared to amyloid-specific antibodies, which are currently the gold standard for labeling these plaques. This technique takes less time and is as equally effective as any of the commonly used amyloid labeling methods. Curcumin strongly binds to amyloid plaques and can be used as an imaging probe during PET scans of the brain and for relatively noninvasive screenings using retinal scanning.Curcumin also binds to neurofibrillary or tau tangle in Alzheimer's disease, alpha synuclein in Parkinson's disease, and mutant Huntington's proteins in Huntington's disease. Demonstrating the procedure with Panchanan Maiti will be Zackary Bowers, a graduate student, and Alexandra Plemmons, a research scientist from my lab. After confirming a lack of response to pain reflex in an anesthetized 12 month old Alzheimer's disease model mouse, place the mouse in the supine position on a surgery tray and make an incision into the abdomen and through the diaphragm.Insert a 21 gauge perfusion needle into the incision and make a small incision at the right auricle to allow the perfusion fluid to drain from the body. Use a gravity fed perfusion system to allow ice cold 0.1 molar PBS fluid to flow into the needle for five to six minutes at a 20 to 25 milliliters per minute flow rate. If the perfusion is performed correctly, a clear liver will be observed.A proper animal perfusion is essential because if the blood is not removed completely, curcumin may bind to the blood vessels and result in a green fluorescent background signal. When all of the PBS has been delivered, switch the buffer valve to an ice cold 4%paraformaldehyde solution for eight to 10 minutes before using scissors and forceps to free the brain from the skull. Then use a spatula to place the brain into a vial of 4%paraformaldehyde at least 10 times the tissue volume for four degrees celsius storage until processing.To obtain sections for cryostat sectioning, sequentially immerse the brain in graded sucrose solutions at four degrees celsius for 24 hours per concentration before using a cryostat at negative 22 degrees celsius to obtain 40 micrometer sections, placing 10 to 20 sections per well in a six well plate containing PBS supplemented with 0.02%azide as they are obtained. When all of the samples have been acquired, store the sections at four degrees celsius until further processing. To obtain sections for paraffin sectioning, dehydrate post-fixed perfused brain tissue with sequential graded alcohol solutions for two hours per concentration, followed by two one hour 100%alcohol immersions and two one hour xylene immersions at room temperature.After the last xylene immersion, penetrate the tissue with a one to one xylene to paraffin solution two times for one hour per incubation at 56 degrees celsius in a glass conical glass covered with aluminum foil before immersing the tissues in 56 degree celsius paraffin for four to six hours. At the end of the incubation, use a rotary microtome at room temperature to obtain five micrometer thick sections placing the sections in a 45 degree celsius tissue water bath as they are collected. For curcumin labeling of amyloid beta plaques in cryostat tissue sections, rinse the sections with PBS three times for five minutes per wash before immersing the sections in 70%ethanol for two minutes at room temperature.While the sections are incubating, dissolve one millimolar of stock curcumin in methanol and dilute the solution to a final working concentration of 10 micromolar with 70%ethanol. At the end of the incubation, immerse the sections in the working curcumin solution for 10 minutes at room temperature at 50 rotations per minute. At the end of the staining period, discard the curcumin solution and wash the sections with three two minute washes in 70%ethanol.After the last wash, transfer the sections to polylysine coated glass slides and mount a cover slip onto each slide with an appropriate organic mounting medium. Then view the sections on a florescence microscope using a 10 or 20x objective and the appropriate excitation and emission filters. For histochemical labeling of deep paraffinized tissue sections, immerse the five micrometer thick sections in xylene two times for five minutes at room temperature per immersion, followed by rehydration with graded one minute alcohol solution immersions.At the end of the incubation, wash the sections with graded alcohol solutions for two minutes per concentration followed by clearing with two five minute xylene immersions. After the second xylene immersion, mount each section on a microscope slide with a cover slip and an appropriate mounting medium for florescence microscopy imaging as demonstrated. For labeling of cryostat section specimens with anti-A-beta antibodies, wash the samples three times with fresh PBS per wash in individual wells of a 12 well plate before blocking any non-specific binding with 10%normal goat serum in PBS and 0.5%triton x-100 for one hour at room temperature.At the end of the incubation, discard the blocking solution and incubate the samples with a-beta specific antibody overnight at four degree celsius and 150 rotations per minute. The next morning, wash the sections with three 10 minute washes in fresh PBS per wash, followed by incubation with an appropriate secondary antibody conjugated with a red fluorophore for one hour at room temperature protected from light. At the end of the incubation, wash the sections three times with PBS followed by one wash with 70%alcohol.After the alcohol wash, incubate the sections with 10 micromolar curcumin for 10 minutes at room temperature followed by three one minute 70%alcohol washes. After the last wash, dehydrate the sections with 90%and 100%alcohol for one minute per concentration and clear the sections two times for five minutes per immersion with fresh xylene per incubation. Then mount and image the sections as demonstrated.For intracellular amyloid beta co-localization, stain the sections with anti amyloid beta antibody followed by staining with curcumin at room temperature and 50 rotations per minute protected from light as demonstrated. At the end of the incubation, counterstain with an appropriate nuclear dye for 10 minutes at room temperature and 50 rotations per minute protected from light followed by three washes in PBS. Then mount and image the sections by fluorescence microscopy as demonstrated.Although increasing the incubation time with curcumin slightly increases the fluorescence intensity of the A beta plaques, the number of observed plaques is not significantly different between one and five minutes of staining time. Curcumin can be used to stain A beta plaques and cryosectioned, paraffin embedded, and human Alzheimer's disease tissue samples. Labeling with A beta specific antibody before curcumin staining reveals that the curcumin is completely co-localized with A beta at the same plaques that bind the antibody.After staining with curcumin labeled A beta oligomers, curcumin co-localization with the oligomers can be observed in A beta plaques. Similarly, curcumin also co-localizes with the A beta specific antibody in intracellular spaces, confirming that the stain can label intracellular A beta. Curcumin labeling is also more prominent than conventional amyloid binding dyes.Curcumin derivatives found in tumeric extracts label A beta plaques comparatively to curcumin in human Alzheimer's disease brain tissue regardless of the type of mounting medium used. In addition, curcumin immunofluorescence signals and counterstaining intensity are maintained even after staining with typical nuclear stains and other cell markers. We met that constant sections should be thinner than 40 microns.This training procedure should be kept under 30 minutes and the optimal curcumin concentration is one to two micromolar. Double leveling retarded biomarkers can be performed on the same tissue and can provide a greater specificity of which types of brain cells are being leveled. Given that curcumin can reduce the amyloid plaque load, the precise mechanisms of how this process occurs is currently being investigated at both the cellular and molecular levels.

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12.0K visualizações.  Ascension St. Mary's Hospital. Este protocolo é o método mais eficiente e econômico para rotular com precisão placas beta-amilóides, em comparação com anticorpos específicos amiloides, que atualmente são o padrão-ouro para rotular essas placas. Esta técnica leva menos tempo e é tão eficaz quanto qualquer um dos métodos de rotulagem amiloide comumente usados. A curcumina se liga fortemente a placas amiloides e pode ser usada como uma sonda de imagem durante tomografias pet do cérebro e para triagens relativam...