This work was supported by grants from the National Natural Science Foundation of China (U1503223, 81673407, 21475131), the Applied Research Project on Nonprofit Technology of Zhejiang Province (2016C37110), the Ningbo International Science and Technology Cooperation Project (2014D10019), the Ningbo Municipal Innovation Team of Life Science and Health (2015C110026), the Ningbo Sci &amp; Tech Project for Common Wealth (2017C50042), the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund, and the K. C. Wong Magna Fund at Ningbo University.

1. Solution Preparation
NOTE: See Table of Materials for reagent sources.
<ol>
	<li>Prepare a 5% bovine serum albumin (BSA) solution by adding 5 g of BSA to 100 mL of double-distilled water. Mix them completely by vortexing them. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-oligomer antibody A11 solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of the 5% BSA solution. Mix them completely by vortexing them. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-A&#946; antibody 6E10 solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare an anti-fibrillar oligomer antibody OC solution (1:1,000) by adding 10 &#181;L of antibody stock solution to 10 mL of 5% BSA solution. Mix them completely by vortexing. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Prepare a Tris-buffered saline (TBS) stock solution by adding 24 g of Tris base and 88 g of NaCl to 1,000 mL of double-distilled water. Adjust the pH to 7.4. Store the solution at 4 &#176;C for up to 3 months.</li>
	<li>Prepare a Tris-buffered saline and Tween-20 (TBST) solution by adding 1 mL of Tween-20 to 100 mL of TBS stock solution and 900 mL double-distilled water.</li>
	<li>Prepare a secondary antibody solution by adding 10 &#181;L of horseradish peroxidase (HRP) Goat anti-Rabbit IgG (H + L) to 10 mL of TBST solution. Mix them completely by vortexing them. Store the solution at 4 &#176;C for up to 1 month.</li>
	<li>Dissolve 3.68 g of curcumin in 1 mL of dimethyl sulfoxide (DMSO) to form a 10-mM curcumin stock solution.</li>
	<li>Dissolve 5 mg of synthetic A&#946;1-42 in 2 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to form a 2.5-mg/mL A&#946; monomer solution. Place it at room temperature (25 &#176;C) for 20 min. Make 100 &#181;L aliquots and store at -20 &#176;C for up to 6 months. 
	NOTE: This procedure should be run as fast as possible. The pipette tips should be cut off to ensure accurate pipetting.</li>
	<li>Prepare electrochemiluminescence (ECL) fluid by mixing ECL fluid A and B with a volume ratio of 1:1. This solution should be prepared just before use.</li>
</ol>
2. Sample Preparation
NOTE: Perform the sample preparation 2 days before the dot blotting analysis.
<ol>
	<li>Add 900 &#181;L of double-distilled water to a tube of A&#946;1-42 monomer solution; the concentration of A&#946;1-42 will be 0.25 mg/mL. Place the mixture at room temperature for 20 min.</li>
	<li>Evaporate the solution with high-purity nitrogen gas until its volume is about 850 &#181;L. The concentration of the A&#946;1-42 solution will be about 0.29 mg/mL. 
	NOTE: The solution should be shaken from time to time to ensure the HFIP is fully evaporated. Normally, after 30 min of evaporation, the volume of the residual solution is about 850 &#181;L.</li>
	<li>Dilute the curcumin stock solution to a curcumin working solution (0.2 and 2 &#181;M) with double-distilled water. Mix the A&#946;1-42solution and curcumin working solution with a volume ratio of 1:1. The final concentrations of curcumin will be 0.1 and 1 &#181;M. 
	NOTE: Potential oligomer inhibitors can be mixed with the A&#946;1-42 solution in any ratio if needed.</li>
	<li>Shake the solution continuously in a magnetic agitator (see Table of Materials).
	<ol>
		<li>Fix a plastic divider box to the magnetic agitator. Place 2 magnetic stir bars at 2 corners of the box and place the sample tubes in the center of the box.</li>
		<li>Shake the box at room temperature (25 &#176;C) for 48 h. 
		NOTE: The speed of the magnetic agitator is around 60 rpm.</li>
	</ol>
	</li>
	<li>Centrifuge the tubes for 15 min at 4 &#176;C and 18,000 x g. Collect the supernatant.</li>
</ol>
3. Dot Blotting Analysis
NOTE: All incubations are performed on a horizontal shaker.
<ol>
	<li>Cut nitrocellulose membrane into 1-cm wide strips. 
	NOTE: The width of the nitrocellulose membrane can be adjusted according to experimental needs.</li>
	<li>Place 2-&#181;L samples evenly on 2 strips (strip 1 and 2) with each point interval at 0.5 cm.</li>
	<li>Place the strips at room temperature for 5 min until the droplet on the band is dry.</li>
	<li>Incubate the strips with a 5% BSA solution for 30 min at room temperature.</li>
	<li>Rinse the strips with a TBST solution for 5 min at room temperature.</li>
	<li>Aspirate the TBST solution. For 1 h at room temperature, incubate strip 1 with an anti-oligomer antibody A11 solution and strip 2 with an anti-A&#946; antibody 6E10 solution.</li>
	<li>Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.</li>
	<li>Aspirate the TBST solution. Incubate the strips with a secondary antibody solution for 40 min at room temperature.</li>
	<li>Rinse the strips with a TBST solution three times, each time for 5 min at room temperature.</li>
	<li>Evenly apply the mixed ECL fluid to the surface of the strips. Expose the strips in an automatic chemiluminescence imaging system (see Table of Materials). 
	NOTE: Normally, 300 &#181;L of ECL fluid is enough for one membrane. The membrane must be kept moist during the exposure. The exposure time is automatically calculated by the imaging system.</li>
	<li>Do a grayscale analysis using ImageJ (National Institutes of Health) or another image-processing software to obtain semi-quantitative results.</li>
</ol>

<ol><li>Lauren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+prion+protein+as+a+therapeutic+target+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Journal+of+Alzheimer%27s+Disease%22%5BJournal%5D)">Cellular prion protein as a therapeutic target in Alzheimer's disease.</a> Journal of Alzheimer's Disease. 38 (2), 227-244 (2014).</li>
<li>De Maio, A., Rivera, I., Cauvi, D. M., Arispe, N. <a target="_blank" href="http://www.cell.com/biophysj/comments/S0006-3495(16)33411-7">Modulation of amyloid peptide oligomerization and toxicity by extracellular Hsp70.</a> Biophysical Journal. 112 (3), 444(2017).</li>
<li>Di Scala, C., Chahinian, H., Yahi, N., Garmy, N., Fantini, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+Alzheimer%27s+beta-amyloid+peptides+with+cholesterol%3A+mechanistic+insights+into+amyloid+pore+formation%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Interaction of Alzheimer's beta-amyloid peptides with cholesterol: mechanistic insights into amyloid pore formation.</a> Biochemistry. 53 (28), 4489-4502 (2014).</li>
<li>Xiang, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fucoxanthin+inhibits+beta-amyloid+assembly+and+attenuates+beta-amyloid+oligomer-induced+cognitive+impairments%5BTitle%5D)+AND+%22Journal+of+Agricultural+and+Food+Chemistry%22%5BJournal%5D)">Fucoxanthin inhibits beta-amyloid assembly and attenuates beta-amyloid oligomer-induced cognitive impairments.</a> Journal of Agricultural and Food Chemistry. 65 (20), 4092-4102 (2017).</li>
<li>Chang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protection+against+beta-amyloid-induced+synaptic+and+memory+impairments+via+altering+beta-amyloid+assembly+by+bis%28heptyl%29-cognitin%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Protection against beta-amyloid-induced synaptic and memory impairments via altering beta-amyloid assembly by bis(heptyl)-cognitin.</a> Scientific Reports. 5, 10256(2015).</li>
<li>Yam, G. H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+amyloid+aggregate+forming+ability+of+TGFBI+mutants+that+cause+corneal+dystrophies%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.</a> Investigative Ophthalmology & Visual Science. 53 (9), 5890-5898 (2012).</li>
<li>Tomaselli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+alpha-to-beta+conformational+transition+of+Alzheimer%27s+A+beta-%281-42%29+peptide+in+aqueous+media+is+reversible%3A+A+step+by+step+conformational+analysis+suggests+the+location+of+beta+conformation+seeding%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">The alpha-to-beta conformational transition of Alzheimer's A beta-(1-42) peptide in aqueous media is reversible: A step by step conformational analysis suggests the location of beta conformation seeding.</a> ChemBioChem. 7 (2), 257-267 (2006).</li>
<li>Shigemitsu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+magnetic+resonance+evidence+for+the+dimer+formation+of+beta+amyloid+peptide+1-42+in+1%2C1%2C1%2C3%2C3%2C3-hexafluoro-2-propanol%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.</a> Analytical Biochemistry. 498, 59-67 (2016).</li>
<li>Khan, M. V., Rabbani, G., Ahmad, E., Khan, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluoroalcohols-induced+modulation+and+amyloid+formation+in+conalbumin%5BTitle%5D)+AND+%22International+Journal+of+Biological+Macromolecules%22%5BJournal%5D)">Fluoroalcohols-induced modulation and amyloid formation in conalbumin.</a> International Journal of Biological Macromolecules. 70, 606-614 (2014).</li>
<li>Fang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((5-hydroxycyclopenicillone%2C+a+new+beta-amyloid+fibrillization+inhibitor+from+a+sponge-derived+fungus+trichoderma+sp+HPQJ-34%5BTitle%5D)+AND+%22Marine+Drugs%22%5BJournal%5D)">5-hydroxycyclopenicillone, a new beta-amyloid fibrillization inhibitor from a sponge-derived fungus trichoderma sp HPQJ-34.</a> Marine Drugs. 15 (8), 260(2017).</li>
<li>Shiao, Y. J., Su, M. H., Lin, H. C., Wu, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Echinacoside+ameliorates+the+memory+impairment+and+cholinergic+deficit+induced+by+amyloid+beta+peptides+via+the+inhibition+of+amyloid+deposition+and+toxicology%5BTitle%5D)+AND+%22Food+%26+Function%22%5BJournal%5D)">Echinacoside ameliorates the memory impairment and cholinergic deficit induced by amyloid beta peptides via the inhibition of amyloid deposition and toxicology.</a> Food & Function. 8 (6), 2283-2294 (2017).</li>
</ol>

The authors have nothing to disclose.

In this protocol, we have reported a method to prepare samples containing A&#946;1-42 oligomer, and to analyze the amounts of A11-positive A&#946;1-42 oligomer by a dot blotting analysis. Although our methods for the preparation of A&#946;1-42 oligomer-rich samples are quite simple, reliable, and reproducible, there are still some points to be noticed. Firstly, HFIP is used to dissolve the synthetic A&#946;1-42 peptide. An aggregated A&#946;1-42 peptide can disassemb...

Alzheimer&#39;s disease (AD) is one of the most important neurodegenerative diseases affecting elderly people worldwide1. It is widely accepted that the abnormal aggregation of &#946;-amyloid (A&#946;) may be the leading pathological factor of AD. A&#946; aggregates are the main components of the senile plaques, one of the biological markers in the brains of AD patients. Moreover, A&#946; aggregates, including oligomers in particular, produce potent neurotoxicity, which might be the cause of neuronal death as AD progresses. Therefore, the inhibition of A&#946; oligomer formation might prevent neurodegeneration, and A&#946; oligomer inhibitors could be developed as disease-modifying treatments of AD. Many studies have used a synthetic A&#946; peptide to form oligomers in vitro, explore morphologies and structures of artificial A&#946; oligomers, and investigate the inhibitors of A&#946; oligomer using in vitro models2,3,4. However, different in vitro formation protocols of A&#946; oligomer could lead to oligomer with different morphological characteristics, which might cause the incomparable results among different research groups. Therefore, a standard formation protocol for A&#946; oligomer is urgently needed.
Until now, not many methods have been reported to directly detect A&#946; oligomers. Transmission electronic microscopy (TEM), non-denaturing gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and dot blotting analysis can be used to examine the amount and/or morphology of A&#946; oligomer in vitro5,6. For example, the morphology and structure of A&#946; oligomer can be observed in TEM. The relative amounts and molecular size of A&#946; aggregations could be measured by non-denaturing gel electrophoresis. ELISA could be used to determine A&#946; oligomer in serum, plasma, and extracts from brain tissue. Lastly, dot blotting analysis, a technique used for detecting, analyzing, and identifying proteins, could be used to evaluate the relative concentration of A&#946; oligomer in different samples with the help of oligomer-specific and A&#946;-specific antibodies. Moreover, a dot blotting assay offers significant time savings, as gel electrophoresis and the blotting procedures for gels are not required. Therefore, this assay is normally used to screen potential A&#946; oligomer inhibitors. The overall goal of this protocol is to describe a relatively simple, reliable, and reproducible method to prepare an A&#946;1-42 oligomer-rich sample, to analyze the amounts of A&#946;1-42 oligomer by dot blotting analysis, and to screen A&#946; oligomer inhibitors using semi-quantitative experimental results.

<table><tbody><tr><td>A11 (ab126892 Rb pAb to Amyloid Oligomers)</td><td>Abcam</td><td>GR91739-58</td><td></td></tr><tr><td>6E10 (beta-Amyloid Rabbit Ab)</td><td>Cell&amp;nbsp;Signalling Technology</td><td>2454S</td><td></td></tr><tr><td>OC (Anti-Amyloid Fibrils OC Antibody)</td><td>Millipore</td><td>AB2286</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP) Marker Goat anti rabbit IgG (H+L)&amp;nbsp;</td><td>Beyotime</td><td>A0208</td><td></td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>GL Biochem</td><td>52487</td><td></td></tr><tr><td>1,1,1,3,3,3-Hexafiuoro-2-propanol</td><td>Aladdin</td><td>I1523078</td><td></td></tr><tr><td>Curcumin</td><td>Sigma</td><td>C1386</td><td></td></tr><tr><td>Albumin Bovine V</td><td>Solarbio</td><td>A8020</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>D920BA0003</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>SCR</td><td>30166428</td><td></td></tr><tr><td>TRIS</td><td>Solarbio</td><td>T8060</td><td></td></tr><tr><td>Glycine</td><td>Solarbio</td><td>G8200</td><td></td></tr><tr><td>&amp;nbsp;Dimethyl sulfoxide</td><td>Solarbio</td><td>D8370</td><td></td></tr><tr><td>5&amp;times;nondenaturing gel PAGE Protein Marker</td><td>Beyotime</td><td>P0016</td><td></td></tr><tr><td>Genshare CFAS anyKD PAGE</td><td>Genshare</td><td>JC-PE022</td><td></td></tr><tr><td>Pure Nitrocellulose Blotting Membrane</td><td>Pall Corporation</td><td>T50189</td><td></td></tr><tr><td>Methanol</td><td>SCR</td><td>10014118</td><td></td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Super low range&amp;nbsp;protein Marker</td><td>Solarbio</td><td>PR1300</td><td></td></tr><tr><td>Transfer Membranes</td><td>Immobilon-PSQ</td><td>ISEQ00010</td><td></td></tr><tr><td>BeyoECL Star</td><td>Beyotime</td><td>P0018A</td><td></td></tr><tr><td>Commassie Blue Fast staining solution</td><td>Beyotime</td><td>P0017</td><td></td></tr><tr><td>All - automatic chemiluminescence imaging system</td><td>Tanon</td><td>Tanon 5200</td><td></td></tr><tr><td>Image J</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Image processing software</td><td>Adobe</td><td>Photoshop CS6</td><td></td></tr><tr><td>Magnetic agitator</td><td>Shanghai Huxi</td><td></td><td></td></tr></tbody></table>

a11-positive &#946;-amyloid oligomer preparation and assessment using dot blotting analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

To investigate whether an A&#946;1-42 monomer can form an A&#946;1-42 oligomer after preparation, TEM analysis was used. No visible aggregates were observed in the HFIP-dissolved A&#946;1-42 monomer sample (Figure 1A). Moreover, mainly globular aggregates with a diameter of around 10 - 80 nm were observed in the A&#946;1-42 sample after 48 h of shaking, suggesting that A&#946;1-42 forms o...

Watch this Scientific Journal Video about A11-positive Î²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis at JoVE.com

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

a11-positive-amyloid-oligomer-preparation-assessment-using-dot

Research

JoVE Journal

Biochemistry

11.8K Views.  Ningbo University. Dit protocol wordt beschreven hoe voor te bereiden Aβ oligomeren van een synthetische peptide in vitro en relatieve hoeveelheid Aβ oligomeren evalueren door een stip bevlekken analyse.

Video: A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neuroscience

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

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.

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

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Biology

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Immunology and Infection

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

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