Authors thank Masanori Kawasaki, Kazuyoshi Aso, and Michael Foley from Tri-Institutional Therapeutics Discovery Institute (TDI), New York for synthesis of A&#946;-fibrinogen interaction inhibitors and their valuable suggestions. Authors also thank members of the Strickland lab for helpful discussion. This work was supported by NIH grant NS104386, the Alzheimer&#39;s Drug Discovery Foundation, and Robertson Therapeutic Development Fund for H.A., NIH grant NS50537, the Tri-Institutional Therapeutics Discovery Institute, Alzheimer&#39;s Drug Discovery Foundation, Rudin Family Foundation, and John A. Herrmann for S.S.

1. Preparation of A&#946;42 and Fibrinogen for Analysis
<ol>
	<li>Prepare monomeric A&#946;42 from lyophilized powder
	<ol>
		<li>Warm A&#946;42 powder to room temperature and spin down at 1,500 x g for 30 s.</li>
		<li>Add 100 &#181;L of ice-cold hexafluoroisopropanol (HFIP) per 0.5 mg of A&#946;42 powder and incubate for 30 min on ice. 
		CAUTION: Use care when handling HFIP and perform all steps in a chemical hood.</li>
		<li>Prepare 20 &#181;L aliquots and let the films air dry in a chemical hood for 2-3 h. Films can be stored at -20 &#176;C.</li>
		<li>Reconstitute monomeric A&#946;42 film in 10 &#181;L of fresh dimethyl sulfoxide (DMSO) by agitation in a bath sonicator for 10 min at room temperature.</li>
		<li>Add 190 &#181;L of 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.4) and pipette up and down gently.</li>
		<li>Remove the protein aggregates by centrifugation at 20,817 x g at 4 &#176;C for 20 min.</li>
		<li>Incubate the supernatant at 4 &#176;C for overnight and next day centrifuge the solution at 20,817 x g at 4 &#176;C for 20 min to discard any further protein aggregates.</li>
		<li>Measure A&#946;42 concentration by bicinchoninic acid (BCA) assay. Serial dilute purified bovine serum albumin (BSA) from 1 mg/mL to 0.0625 mg/mL to produce a protein standard, add 10 &#181;L of each standard to the wells of a multi-well plate in triplicate. Dilute A&#946; samples 1:4 and add 10 &#181;L to the wells in triplicate. Mix BCA solutions A and B and add 200 &#181;L to each well. Incubate for 30 min at 37 &#176;C and read on a plate reader at 562 nm. Keep the remaining A&#946; solution on ice and use this preparation for turbidity assay and SEM.</li>
	</ol>
	</li>
	<li>Prepare Fibrinogen solution
	<ol>
		<li>Measure 20 mg of lyophilized fibrinogen powder into a 15 mL tube and re-suspend with pre-warmed 2 mL of 20 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES) buffer (pH 7.4).</li>
		<li>Incubate in a 37 &#176;C water bath for 10 min.</li>
		<li>Filter solution through a 0.2 &#181;m syringe filter and store at 4 &#176;C for 30 min. Take out the solution from 4 &#176;C and filter again through 0.1 &#181;m syringe filter to remove fibrinogen aggregates or pre-existing fibrin.</li>
		<li>Measure the fibrinogen concentration by BCA assay. Follow the instructions from Step 1.1.8. Keep the remaining fibrinogen solution at 4 &#176;C and use this preparation for turbidity assay and SEM.</li>
	</ol>
	</li>
</ol>
2. Clot Turbidity Assay
<ol>
	<li>To each experimental well of the 96-well plate, add 20 &#181;L of 30 &#181;M A&#946;42 solution from Step 1.1.8 so that its final concentration is 3.0 &#181;M in 200 &#181;L of the buffer. Add the same volume of 5% DMSO in 50 mM Tris buffer (pH 7.4) to buffer control wells in the 96-well plate. 
	NOTE: The exact volume of A&#946;42 at this step is not important. For low concentration preparations, a higher volume can be used, and the volume of the clot formation buffer can be adjusted. The final volume should remain 200 &#181;L.</li>
	<li>If testing inhibitory compounds, dilute the compound to the working concentration as DMSO. Add compound or DMSO alone for a control to the wells with A&#946;42 and mix well. 
	NOTE: A&#946;42 solution should form a discrete droplet at the bottom of the well, the compound or DMSO should be pipetted directly into the center of the droplet.</li>
	<li>Dilute the fibrinogen stock solution from Step 1.2.4 in clot formation buffer (20 mM HEPES buffer (pH 7.4), 5 mM CaCl2, and 137 mM NaCl) and add it to A&#946;42 containing and control wells of the 96-well plate. Adjust the volume of the fibrinogen solution so that its final concentration becomes 1.5 &#181;M in total 200 &#181;L reaction volume. Pipette the solution slowly and avoid forming bubbles. Incubate the plate at room temperature for 30 min shaking on a rotating platform. 
	NOTE: The volume of fibrinogen solution may vary depending on its stock concentration, but the total volume in each well should be 170 &#181;L while incubation.</li>
	<li>Prepare thrombin solution by dissolving commercially purified thrombin powder in ddH2O to make a stock solution of 50 U/mL. Dilute to the 5 U/mL working solution in 20 mM HEPES buffer (pH 7.4) directly before use.</li>
	<li>After 30 min of incubation, simultaneously add 30 &#181;L of thrombin solution (5 U/mL) into the fibrinogen solutions in the 96-well plate from Step 2.3 using multichannel pipette. Addition of thrombin will immediately initiate the clot formation. Therefore, add thrombin directly into the center of the fibrinogen solution with care to avoid forming bubbles. 
	NOTE: The activity of thrombin can vary between experimental conditions, as well as thrombin lots. The correct concentration required to robustly produce clots may have to be determined empirically.</li>
	<li>Read the absorbance of in vitro clot on a plate reader immediately following the thrombin addition. Measure the absorbance at 350 nm over the course of 10 min, every 30-60 s. Perform the entire assay at room temperature. 
	NOTE: Some inhibitor compounds may alter the solution absorbance at 350 nm, in which case the turbidity can be measured at 405 nm.</li>
</ol>
3. Scanning Electron Microscopy
<ol>
	<li>Preparation of clot, fixation, and washing
	<ol>
		<li>Place clean siliconized glass circle cover slides (12 mm) into a 12-well or 24-well plate using forceps.</li>
		<li>Prepare fibrinogen in clot formation buffer. See protocol step 1.2 for details.</li>
		<li>To evaluate the effect of A&#946;-fibrinogen interaction inhibitors on fibrin clot structure, follow the instructions for incubation as described for the turbidity assay (Step 2). Always include control wells which contain fibrinogen in the absence of both A&#946; and inhibitors.</li>
		<li>Pipette 80 &#181;L of the fibrinogen mixture from Step 3.1.3 on the cover slides. Gently spread the solution so that it is evenly distributed on the cover slide.</li>
		<li>Dilute thrombin stock (50 U/mL) into 20 mM HEPES buffered saline to a final concentration of 2.5 U/mL and add 20 &#181;L directly to the center of to the fibrinogen solution without mixing. 
		NOTE: If required, a higher thrombin working concentration (5 U/mL) can be used.</li>
		<li>Cover the 12-well plate with a plastic lid and leave it at room temperature for 30-60 min.</li>
		<li>While clotting reaction is running, prepare dehydration and fixation solutions. Prepare sodium cacodylate buffer (0.1 M, pH 7.4). Dilute 10% glutaraldehyde stock solution in sodium cacodylate buffer (0.1 M, pH 7.4) to make a 2% working solution of glutaraldehyde. Keep all these solutions on ice. Freshly diluted glutaraldehyde (2%) should be used within 1 week, when properly stored in a refrigerator at 4 &#176;C.</li>
		<li>Dilute absolute ethanol (100%) in double distilled water (80%, 50%, and 20% ethanol). Keep these ethanol solutions and absolute ethanol (100%) on ice. 
		&#8203;CAUTION: Use care when handling sodium cacodylate and glutaraldehyde, perform these steps in a chemical hood.</li>
		<li>After 30 min, gently wash the clots with ice-cold sodium cacodylate buffer (0.1 M) twice. Add 2 mL of the buffer to each well such that the clot is completely submerged. Cover the well plate with a lid and leave it for 2 min at room temperature. After 2 min, gently remove the buffer using a 1 mL pipette or narrow stem transfer pipette. Repeat once.</li>
		<li>Fix the clots with ice-cold glutaraldehyde (2%). Keeping the well plate on ice, add around 2-3 mL of 2% glutaraldehyde to each well. Make sure the clots are completely submerged. Cover and leave the plate on ice for 30 min.</li>
		<li>After 30 min, gently remove the glutaraldehyde from each well and wash the clots using sodium cacodylate buffer (0.1 M) as described above (2 min, twice). Keep the well plate on ice.</li>
	</ol>
	</li>
	<li>Serial dehydration of clot and critical point drying
	<ol>
		<li>Dehydrate the clots in a graded series of ice-cold ethanol washes prepared in Step 3.1.8 (20%, 50%, 80%, 100%, and again100%) for 5 min each. For each ethanol series, add 2-3 mL, making sure the clots are submerged. Cover and incubate on ice.</li>
		<li>After each ethanol step, remove the ethanol solution using a 1 mL pipette or disposable pipette dropper. Do not remove the ethanol completely. Make sure the clot surface is not exposed to air. 
		NOTE: Dehydration in absolute ethanol (100%) should be performed twice.</li>
		<li>While keeping the sample submerged in the final 100% ethanol, transfer to a critical point dryer (CPD). Use a CPD sample holder or a cover slip holder with a washer for transferring the slides into the CPD chamber. Place at least one washer between each slide.</li>
		<li>Take out the cover slide from the CPD chamber and mount it on an SEM stub using carbon tape.</li>
	</ol>
	</li>
	<li>Sputter coating and imaging
	<ol>
		<li>Transfer all the samples with SEM stub to the sputter coating chamber.</li>
		<li>Sputter coat less than 20 nm of gold/palladium or other conductive materials, such as carbon, using a vacuum sputter coater. 
		NOTE: We have used 18 nm of gold/palladium coating. The sputter coating was performed for 45 s and the coating speed was 4 &#197;/ s. Sputter coated samples are stable when kept properly in a dry environment at room temperature for few weeks. SEM analysis can be performed anytime.</li>
		<li>Acquire images on a scanning electron microscope equipped with the SE2 detector at 4 kV. 
		NOTE: The image pixel size in this image set range from 13 nm- 31 nm.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The methods described here provide a reproducible and rapid means of assessing fibrin clot formation in vitro. Furthermore, the simplicity of the system makes the interpretation of how A&#946; affects the fibrin clot formation and structure relatively straight-forward. In this lab&#39;s previous publication, it was shown that these assays can be used to test compounds for their ability to inhibit the A&#946;-fibrinogen interaction13,14. Using these two assays, a series of synthesized compounds were analyzed for the ability to inhibit the A&#946;-fibrinogen interaction. Actually, the clot turbidity assay can be used to narrow the pool of hit compounds to those which have therapeutic potential, as not all compounds that can inhibit the A&#946;-fibrinogen interaction can also restore fibrin clot formation.
There are a few aspects of the turbidity assay that may require troubleshooting or limit the uses of this technique. The major limitation is that there can be some variations between the experiments that may complicate the interpretation of the results. Some of this variation is to be due to the thrombin activity. To troubleshoot for thrombin activity, users can test multiple concentrations of thrombin for clot-formation activity prior to beginning experimentation with A&#946; and test compounds. Under the experimental conditions presented here, A&#946;, in the absence of fibrinogen, does not increase the turbidity of the solution above background levels indicating that observed turbidity curves are due to fibrin polymerization. However, A&#946; is an aggregation-prone peptide and a higher concentration of A&#946; solution when kept for a very long time at room temperature or 37 &#176;C (several hours) can form fibrillar aggregates which may result in increased turbidity. If analyzing the effect an A&#946; solution that has been stored for long periods of time at room temperature or warmer, the aggregation status of the solution can be determined by transmission electron microscopy. In the protocol described here, freshly prepared A&#946; and fibrinogen solutions are used, which should eliminate aggregation issues. However, to ensure the quality of these preparations they have been assessed by transmission electron microscopy. Additionally, when using a new lot of commercial A&#946; peptide, the extent of oligomerization and quantity of oligomers should be assessed by transmission electron microscopy. Because there might be lot to lot variation in peptide quality and ratio of preformed aggregates and monomeric A&#946;, which certainly affects the oligomerization rate. It is advisable to check the concentration of A&#946; solution (BCA method) before incubating for oligomerization. To minimize these variations, we tried using at least 0.1 mg/mL of A&#946; solution for oligomer formation reaction.
Because this assay is also sensitive to small environmental changes such as temperature and motion, turbidity readings from different experiments should not be analyzed together. This means that the number of conditions that can be compared is limited by the number of wells that thrombin can be simultaneously added to with a multichannel pipette (i.e., 12). Users should also be aware that viscosity or color in the buffers and test compounds can lead to a high background turbidity, obscuring the signal from the fibrin clot and making interpretation of the experiment difficult. However, if all of the controls are included in each experiment, it should be possible to readily determine if background signal is altering the turbidity absorbance.
The turbidity assay provides information about whether formation of the fibrin clot is hindered by A&#946; and furthermore if inhibitor compounds can alleviate this effect. However, it does not reveal how the structure of the fibrin clot is altered in response to A&#946; and/or hit compounds. This question can be addressed by SEM, as this allows for direct visualization of the clot architecture. SEM is a well-established technique and is routinely used for visualizing clot structure from purified fibrinogen or from plasma. However, the traditional clot preparation process for SEM analysis can be complicated and time-consuming. Furthermore, small differences in protocols between different research groups can make it challenging to replicate results24. To address these issues this optimized protocol was developed, with which the effect of A&#946; to induce thinner fibrin strands and clumps of protein can be observed (Figure 3). As seen with the turbidity assay, TDI-2760 alleviates the effect of A&#946;, restoring the typical structure of fibrin bundles (Figure 3).
There are a few steps in the SEM sample preparation, which may also require troubleshooting. When using very low concentrations of fibrinogen (0.5 &#181;M or less), the clots may not be firmly attached to the glass slide and can be damaged/washed away during the fixation/washing steps. If using low concentrations of fibrinogen, the clot formation time, between the addition of thrombin and fixation, can be increased up to several hours, as this may increase the clot stability. Also, users should avoid using high strength phosphate buffer or phosphate buffered saline for both the turbidity and SEM assays, as phosphate ions may interfere with calcium-mediated clot formation process. If using any other high salt containing buffer for clot formation and SEM analysis, after fixation, washing steps should also be performed with cold ddH2O.
SEM analysis of non-conductive samples such as fibrin clots requires coating with charged particles such as, gold, palladium, silver or carbon. Coating thickness is an important factor in SEM imaging, as it is possible that a very thick metal coating can mask the ultra-structure surface topography of the fibrin clot. In this protocol, thin gold/palladium coating (less than 20 nm) are used for sputter coating. To achieve less than 20 nm thickness, the sputtering was done for 45 s with a coating rate of 4 &#197;/s. This thin coating (18 nm) does not appear to mask the surface features of fibrin assembly. However, if concerned about masking the ultra-structure of the clot, carbon coating can be used as an alternative to gold/palladium, as it will leave less &#34;islets&#34; of coating molecules on the material.
While the focus here has been on the A&#946;-fibrinogen interaction, this protocol can be readily modified to analyze the interaction of other proteins or compounds with the fibrin clot. Following these instructions, investigators should be able to reproduce in vitro fibrin clot formation and perform analysis with these streamlined clot-turbidity assay and SEM protocols. As shown here with the previously published inhibitor compound TDI-2760, these methods provide valuable information regarding fibrin clot formation that can be applied to further studies both in vitro and in vivo.

Alzheimer's disease (AD), a neurodegenerative disease leading to cognitive decline in elderly patients, predominately arises from abnormal beta-amyloid (A&#946;) expression, aggregation and impaired clearance resulting in neurotoxicity1,2. Despite the well-characterized association between A&#946; aggregates and AD3, the precise mechanisms underlying the disease pathology are not well understood4. Increasing evidence suggests that neurovascular deficits play a role in the progression and severity of AD5, as A&#946; directly interacts with the components of the circulatory system6. A&#946; has a high-affinity interaction with fibrinogen7,8, which also localizes to A&#946; deposits in both AD patients and mouse models9,10,11. Furthermore, the A&#946;-fibrinogen interaction induces abnormal fibrin-clot formation and structure, as well as resistance to fibrinolysis9,12. One therapeutic possibility in treating AD, is alleviating circulatory deficits by inhibiting the interaction between A&#946; and fibrinogen13,14. We, therefore, identified several small compounds inhibiting the A&#946;-fibrinogen interaction using high throughput screening and medicinal chemistry approaches13,14. To test the efficacy of A&#946;-fibrinogen interaction inhibitors, we optimized two methods for the analysis of in vitro fibrin clot formation: clot turbidity assay and scanning electron microscopy (SEM)14.
Clot turbidity assay is a straight-forward and rapid method for monitoring fibrin clot formation using UV-visible spectroscopy. As the fibrin clot forms, light is increasingly scattered and the turbidity of the solution increases. Conversely, when A&#946; is present, the structure of the fibrin clot is altered, and the turbidity of the mixture is reduced (Figure 1). The effect of inhibitory compounds can be assessed for the potential to restore clot turbidity from A&#946;-induced abnormalities. While the turbidity assay allows for rapid analysis of multiple conditions, it provides limited information on the clot shape and structure. SEM, in which the topography of solid objects is revealed by electron probe, allows for the analysis of the 3D architecture of the clot15,16,17,18 and the assessment of how the presence of A&#946; and/or inhibitory compounds alters that structure9,14. Both spectrometry and SEM are classical laboratory techniques that have been used for various purposes, for example, spectrophotometry is used for monitoring amyloid aggregation19,20. Similarly, SEM is also used to analyze fibrin clot formed from the plasma of Alzheimer's, Parkinson's and thromboembolic stroke patients21,22,23. The protocols presented here are optimized for assessing fibrin-clot formation in a reproducible and rapid manner. 
The following protocol provides the instructions for the preparation of an in vitro fibrin-clot both with and without A&#946;. It also details the methods to analyze the effect of A&#946; on fibrin clot formation and structure. The effectiveness of these two methods for measuring the inhibition of the A&#946;-fibrinogen interaction is demonstrated using TDI-2760, a small inhibitory compound14. These methods, both individually and together, allow for rapid and straightforward analysis of in vitro fibrin clot formation.

<table>
	<tbody>
		<tr>
			<td>Fibriogen, Plasminogen-Depleted, Plasma</td>
			<td>EMD Millipore</td>
			<td>341578</td>
			<td>keep lid parafilm wrapped to avoid exposure to moisture</td>
		</tr>
		<tr>
			<td>Beta-Amyloid (1-42), Human</td>
			<td>Anaspec</td>
			<td>AS-20276</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99%</td>
			<td>Sigma-Aldrich</td>
			<td>105228</td>
			<td></td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE (DMSO), STERILE-FILTERED</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fischer Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer Scientific</td>
			<td>BP310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fischer Scientific</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fischer Scientific</td>
			<td>C70</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Syringe, 0.2&#181;M, 25mm</td>
			<td>Pall</td>
			<td>4612</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia.</td>
			<td>Millipore</td>
			<td>SLVV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid 96 Well Plates High Binding Certified Flat Bottom</td>
			<td>Fischer Scientific</td>
			<td>21377203</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax Plus384</td>
			<td>Molecular Devices</td>
			<td>89212-396</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, 5417R</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 200 Ultrasonic Cleaner</td>
			<td>Fischer Scientific</td>
			<td>15-337-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Rotator</td>
			<td>Thermo Scientific</td>
			<td>2314-1CEQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Spare washers for cover slip holder</td>
			<td>Tousimis</td>
			<td>8766-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Stem Pipets - Sedi-Pet</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>70967-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample holder for CPD (Cover slip holder)</td>
			<td>Tousimis</td>
			<td>8766</td>
			<td></td>
		</tr>
		<tr>
			<td>Mount Holder Box, Pin Type</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>76610</td>
			<td></td>
		</tr>
		<tr>
			<td>Round glass cover slides (12 mm)</td>
			<td>Hampton Research</td>
			<td>HR3-277</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Glutaraldehyde</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>16120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Falcon</td>
			<td>3047</td>
			<td></td>
		</tr>
		<tr>
			<td>Na Cacodylate</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM Stubs, Tapered end pin.</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>75192</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Tabs, Carbon Conductive Tabs, 12mm OD</td>
			<td>Ted Pella</td>
			<td>16084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosamdri-815 Critical Point Dryer with Gold/Palladium target</td>
			<td>Tousimis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Denton Desk IV Coater</td>
			<td>Denton Vacuum</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leo 1550 FE-SEM</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Smart SEM Software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the in vitro clotting (turbidity) assay, the enzyme thrombin cleaves fibrinogen, resulting in the formation of the fibrin network24. This fibrin clot formation causes scattering of the light passing through the solution, resulting in increased turbidity (Figure 1), plateauing before the end of the reading period (Figure 2, green). When the fibrinogen was incubated in the presence of A&#946;42, the turbidity of the solution decreased, with the curve reaching a maximum height of roughly half that of fibrinogen alone (Figure 2A, blue). In a recent publication, a series of A&#946; aggregation blockers were synthesized and assessed for their ability to inhibit the A&#946;-fibrinogen interaction, identifying the compound TDI-276014. We have used TDI-2760 in our study to block A&#946;-fibrinogen interaction. As reported previously, in the presence of TDI-2760, the effect of A&#946; was ameliorated, as the turbidity was higher than with A&#946; alone (Figure 2A, red). The effect of TDI-2760 does not appear to be due to background turbidity as the compound did not change the fibrin clot turbidity when A&#946; was absent (Figure 2A, purple). The in vitro clotting turbidity assay described here is a rapid and simple method by which fibrin-clot formation and factors that may attenuate that process can be observed. GPRP, which is known to interfere with fibrin polymerization via interfering with fibrin monomer knob-hole interactions18,25 can be used as a positive control for the turbidity assay (Figure 2B). Consistent with the earlier reports25, with the presence of GPRP peptide, the fibrin clot turbidity was significantly reduced as compared to the fibrin clot formation with its absence (Figure 2B, blue vs green).
Following the same protocol and conditions as the turbidity assay described above, fibrin clots were prepared in the presence and absence of A&#946; and/or TDI-2760. The clots were then processed for electron microscopy by fixation, dehydration, critical point drying, and gold-sputter coating (Figure 1). Fibrinogen in the presence of only thrombin and CaCl2 formed a fibrin mesh, with elongated and intercalated threads of fibrin as well as larger bundles (Figure 3A). When A&#946; was present, the fibrin threads became thinner, with several sticky clumps/aggregates indicating A&#946;-induced structural abnormalities (Figure 3B). Consistent with the turbidity assay results, TDI-2760 partially restored the structure of the fibrin clot from A&#946;-induced changes, as fewer clumps were present (Figure 3C). Together with the turbidity assay, SEM reveals the extent and quality of A&#946;-induced changes to fibrin clot formation, as well as the effectiveness of an inhibitory compound, TDI-2760.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58475/58475fig1.jpg" /> 
Figure 1: Schematic representation of fibrin clot analysis by turbidity assay and SEM. The&#160;schematic shows the steps involved in fibrin clot analysis and the effect of A&#946; on fibrin clot turbidity and structural topography. The scale bars of SEM images (bottom left) are 2 &#181;m and the magnification is 10,000X. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58475/58475fig2.jpg" /> 
Figure 2: Measurement of the effect A&#946;42 on in vitro fibrin clot formation by turbidity assay. (A) Fibrinogen was incubated in the presence and absence of A&#946;42. Clot formation was induced by thrombin, resulting in increased turbidity of the solution (green). In the presence of A&#946;42, the fibrin clot was abnormally structured, resulting in decreased turbidity (blue). The compound TDI-2760 or DMSO were incubated with the fibrinogen solution, both with and without A&#946;42. TDI-2760 restored A&#946;42-induced decrease of turbidity (red) without altering the normal clot formation (purple). (B) The turbidity of a known fibrinolysis inhibitor, GPRP was also measured as a positive control for this assay. Fibrinogen was incubated in the presence and absence of 1.5 &#181;M GPRP and the turbidity measured after adding thrombin. Similar to A&#946;42, the turbidity of fibrin clot formation was significantly reduced in the presence of GPRP (blue versus green). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58475/58475fig3.jpg" /> 
Figure 3: Scanning electron micrographs of A&#946;-induced abnormalities to fibrin clot architecture. Scanning electron micrographs of fibrin clot structure obtained from purified fibrinogen (A), fibrinogen + A&#946;42 (B), fibrinogen + A&#946;42+TDI-2760 (C). Clot formation was initiated by adding thrombin and CaCl2 to the mixture. The structural analysis revealed that clots formed in the presence of A&#946;42 are thinner and abnormally clumped compared to fibrinogen by itself. TDI-2760, which is known to inhibit A&#946;42-fibrinogen interaction, partially corrected the A&#946;42 induced structural abnormality of the fibrin clot. Scale bar is 1 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58475/58475fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of ÃŽÂ²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy at JoVE.com

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

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 ...

analysis-amyloid-induced-abnormalities-on-fibrin-clot-structure

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9.1K Views.  Rockefeller University. 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.

Video: Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 &#197;, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Negative stain EM is a powerful technique for visualizing macromolecular structure, but different staining techniques can produce varying results in a sample dependent manner. Here several negative staining approaches are described in detail to provide an initial workflow for tackling the visualization of challenging systems.

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...