This research was sponsored by an intramural grant awarded for A.A. This work was supported by a CTSA grant from NCATS awarded to the University of Kansas Medical Center for Frontiers: The Heartland Institute for Clinical and Translational Research (#UL1TR606381). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or NCATS. We are thankful for The University of Kansas Medical Center ALS clinic personal for obtaining IRB approval for blood sample collection from healthy volunteer and ALS patients. The authors thank Emre Agbas for proofreading of the manuscript.

All protocols concerning the processing of human platelets follow the guidelines of both the University of Kansas Medical Center and Kansas City University of Medicine and Biosciences IRB committees.
1. Preparation of buffers and reagents
NOTE: Prepare all samples as per the manufacturer&#39;s guidelines. Wear personal protection equipment (lab coats, gloves, and goggles) during this procedure.
<ol>
	<li>Prepare citrate wash buffer by combining 0.941 g of sucrose (11 mM final concentration), 6.4 mL of 5 M NaCl (128 mM final), 5.4 mL of 0.2 M NaH2PO4 (4.3 mM final), 9.4 mL of 0.2 M Na2PHO4 (7.5 mM final), 0.352 g of sodium citrate (4.8 mM final), and 0.115 g of citric acid (2.4 mM final). Adjust the total volume to 250 mL with ddH2O. Filter through a 0.45 &#181;m filter disk and adjust the pH to 6.5. Store up to 1 year at 4 &#730;C. Bring the solution room temperature (RT) before use22.</li>
	<li>Prepare the rupture buffer by combining 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-Cl (pH 7.4) in a 100 mL final volume. Store up to 1 year at 4 &#730;C. Add 2 &#181;L of phosphatase inhibitors cocktail (1:1000 final) and 1 &#181;L of protease inhibitors cocktail (1:2000 final) into 2 mL of rupture buffer. Keep on ice until use. Discard the unused rupture buffer.</li>
</ol>
2. Platelet isolation
<ol>
	<li>Collect 8&#8211;10 mL of human blood in yellow-cap blood collection tube containing acid-citrate-dextrose (ACD) solution (75 mM trisodium citrate, 124 mM dextrose, and 38 mM citric acid, pH = 7.4; ACD:blood = 1:9). Gently mix the tube content 5x&#8211;6x inverting by hand.</li>
	<li>Centrifuge the tubes at 200 x g in a swinging bucket rotor for 20 min at RT.</li>
	<li>Collect platelet-rich plasma (PRP) (~3&#8211;4 mL) into a 15 mL conical bottom tube and leave approximately 0.5 mL of the PRP from the buffy coat (hazy-looking fraction) to avoid contamination. If any red blood cell contamination occurs, repeat this step.</li>
	<li>Centrifuge the PRP samples at 1,200 x g for 15 min at RT.</li>
	<li>Wash the platelet pellets (P1) by gentle resuspension in 1 mL of citrate wash buffer and pellet by centrifugation at 1,200 x g for 15 min at RT.</li>
	<li>Save the pure platelet pellet. Discard the supernatant.</li>
	<li>Resuspend the platelet pellets in 600 &#956;L of the rupture buffer containing inhibitor cocktails.</li>
	<li>Sonicate the platelet suspension using a sonicator. Place the sample in a mini ice bucket. Set the sonicator at setting 3 for 20 s in continuous mode. 
	NOTE: Make sure to clean the probe with 10% bleach followed by distilled water.</li>
	<li>Centrifuge the sonicated samples at 20,000 x g for 30 min at 4 &#176;C to remove membranous fractions. Aliquot supernatants in 60 &#956;L and store at -80 &#730;C. Avoid repeated thawing/freezing cycles for the platelet cytosolic fractions.</li>
</ol>
3. Preparation for CEI
NOTE: 100 &#181;L of human platelet lysate was combined from ALS patients (n = 8&#8211;10), and healthy subjects (n = 8&#8211;10) were separately pooled and used for the assay optimization.
<ol>
	<li>Fill out in-house generated templates for the CEI layout (Table 1) and sample preparation (Table 2). The sample mixture preparation table is dynamic and will automatically calculate how much volume needs to be removed from source. 
	NOTE: When required source volume entered in dynamic Table-2, 0.1 X sample buffer volume will be automatically calculated.</li>
	<li>Pre-label 25 0.2 mL PCR tubes with capillaries #1&#8211;#25 and place them in a PCR rack. Set on ice.</li>
	<li>Pre-label 0.6 mL microcentrifuge tubes: one for each primary antibody and dilution (if needed) to be used, one for the 0.1x sample buffer, one for the luminol-S/peroxide, and one for each sample to be diluted (if needed). Place them on ice in tube rack.</li>
	<li>Take out the sample buffer, wash buffer, one plate, and a cartridge provided in the CEI separation 12&#8211;230 kDa master kit separation module.</li>
	<li>From the 4 &#176;C refrigerator, take out the antibody dilution buffer, primary antibodies, secondary antibodies, luminol, hydrogen peroxide, and standard pack. Place all reagents on ice, except the standard pack, which remains at RT. 
	NOTE: The reagents from the standard packs are lyophilized and sealed with a foil cover. These should be spun down briefly using a mini centrifuge before opening to reduce product loss. To open, the reagent tubes can either be pierced by a pipette tip or pulled back from the corner.</li>
	<li>To prepare the 400 mM DTT, add 40 &#181;L of deionized water to the clear tube containing the DTT.</li>
	<li>To prepare 40 &#181;L of fluorescent 5x master mix, add 20 &#181;L of the 10x sample buffer and 20 &#181;L of the prepared 400 mM DTT solution to the pink tube provided in kit.</li>
	<li>To prepare the biotinylated ladder, add 16 &#181;L of deionized water, 2 &#181;L of 10x sample buffer, and 2 &#181;L of the prepared 400 mM DTT solution to the white tube provided in kit. Mix gently and transfer into a 0.2 mL PCR tube for denaturing.</li>
	<li>Prepare 0.1x sample buffer by adding 1.5 &#181;L of 10x sample buffer and 148.5 &#181;L of deionized water to a 0.6 mL micro-centrifuge tube. Vortex to mix and place on ice.</li>
	<li>Prepare the desired antibody dilutions. Add antibody diluent in volumes designated to each pre-labeled micro-centrifuge tube. If volumes are identical, use reverse pipetting technique23; if not, pre-rinse the pipette tip before dispensing. 
	NOTE: In this assay, a-TDP-43 pan antibody and a-p(S409/410-2) TDP-43 antibody were used. Anti-ERK antibody was used for an internal control to make sure that assay components are working.</li>
	<li>Perform the reverse pipetting for antibody dilution as described below. Alternatively, additional information can be found in the literature24. 
	NOTE: Reverse pipetting technique is preferred when dispensing small sequential volumes of solutions23. This technique offers some advantages: (i) providing a precise volume, (ii) eliminating the reagent foaming in the tip orifice, and (iii) ideal for small volume (&#60;5 &#181;L) reagents, viscous solutions, surfactant solutions, and solutions with high vapor pressure.
	<ol>
		<li>Put a proper tip in a pipette and press the plunger down to the second stop (Step-2). Immerse the pipet tip a few millimeters into the solution. Slowly release the plunger to fill up the pipet tip with the solution while the tip is still immersed in the solution. Remove the tip from the solution and gently touch against the edge of the reagent reservoir so that excess liquid remaining on the outside of the tip is removed.</li>
		<li>Dispense the solution by pressing the plunger down to first stop (Step-1). Do not dispense the remaining solution in the tip.</li>
		<li>Empty the remaining solution in the tip to the reagent reservoir by pressing the plunger to the second stop (Step-2). Release the plunger to the ready position for the next pipetting step.</li>
		<li>Add the required antibody in volumes designated to each pre-labeled microcentrifuge tube (Table 1) Do not pre-rinse the pipet tip: add it directly to the diluent and flush the tip multiple times to remove antibody. Place the tubes on ice.</li>
	</ol>
	</li>
	<li>To prepare the CEI sample-mix, perform the steps listed below for PCR tubes labeled cap#2 through cap#25: This is in the same order as it appears in Table 1.
	<ol>
		<li>Open all tubes, add 1.6 &#181;L aliquots of fluorescent 5x sample buffer to each tube using a reverse pipetting technique, then close each PCR tube upon the addition of the 5x buffer to minimize sample loss.</li>
		<li>Open all tubes, add 0.1x sample buffer in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use a reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.</li>
		<li>Open all tubes, add protein sample in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.</li>
		<li>Briefly centrifuge all PCR tubes in a benchtop centrifuge (13,000 x g for 30 s), flick/vortex PCR tubes to mix, then repeat the centrifugation.</li>
		<li>Transfer all PCR tubes into thermocycler with a heated lid. Denature samples at defined temperature and duration (i.e., 95 &#730;C for 5 min; 70 &#730;C for 10 min). 
		NOTE: The denaturation temperature and duration need to be optimized for target protein.</li>
		<li>Repeat step 3.12.4.</li>
		<li>Return all PCR tubes to tube rack and place on ice.</li>
		<li>During the denaturing step, prepare the development solution (1:1 luminol-S:peroxide solution), then add 200 &#181;L of luminol-S and 200 &#181;L of peroxide. Place on ice.</li>
		<li>To load a CEI pre-filled plate with the sample prepared above, dispense reagents and samples into the assay plate shown in the assay layout (Figure 1). Avoid introducing air bubbles. 
		NOTE: If volumes and solution are identical, use a reverse pipetting technique. If not, pre-rinse the pipet tip before dispensing and do not expel the remainder into the plate well using the second tab stop on the pipette. A 12&#8211;230 kDa separation module may contain a color-coded plate-loading guide. Place this guide under the plate while adding reagents and samples to the well, which visually helps when sample loading. The plate-loading guide can be downloaded from company website, as well.
		<ol>
			<li>In row E, add 15 &#181;L of luminol:peroxide mix to each well. 
			NOTE: Ideally, prepare this reagent just before use and add to each well. If this is not convenient, this mixture may be prepared no more than 30 min prior to plate loading.</li>
			<li>In row D, to well D1, add 10 &#181;L of streptavidin-HRP.</li>
			<li>In row D, to wells D2&#8211;D25, add 10 &#181;L of designated secondary antibody.</li>
			<li>In row B, to each well, add 10 &#181;L of antibody diluent.</li>
			<li>In row C, to well C1, add 10 &#181;L of antibody diluent.</li>
			<li>In row C, to wells C2&#8211;C25, add 10 &#181;L of designated primary antibody.</li>
			<li>In row A, to well A1, add 5 &#181;L of biotinylated ladder from PCR tube #1.</li>
			<li>In row A, to wells A2&#8211;A25, add 3 &#181;L of the sample, PCR tubes #2&#8211;#25 into corresponding wells #2&#8211;#25.</li>
			<li>Add 500 &#181;L of wash buffer to each designated wash buffer well.</li>
			<li>Centrifuge the plate for 5 min at 1,000 x g at RT.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60638/60638fig01v2.jpg" /> 
Figure 1: Assay layout. Both primary antibody and target protein sample optimization can be performed in one assay. Capillaries 2&#8211;7, 8&#8211;13, 14&#8211;19, and 20&#8211;24 represent various protein concentration and primary antibody range. Capillary 25 represents positive control. Anti-ERK antibody was used; however, any appropriate positive control can be included. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Performing the CEI on plate 1
<ol>
	<li>First, turn on the CEI (Table of Materials) analyzer, then turn on the computer. Open the software (Table of Materials)</li>
	<li>Connect the analyzer to the online system (Table of Materials). This is a necessary step to collect the run data for trouble shooting purposes and data recovery.</li>
	<li>Click on Instrument from the top-left menu, then click Connect. Select the instrument serial number which appears as a pop-up menu. Click Connect.</li>
	<li>Select the &#34;Assay&#34; tab and select New Assay or select a saved template.</li>
	<li>Input assay parameters (Table 1) or modify template currently. Save the file name and location.</li>
	<li>Make sure that the blinking blue color indicator in the analyzer remains solid blue.</li>
	<li>Touch the silver metal button on top of the orange door to open.</li>
	<li>Carefully remove the capillary cartridge from its packaging. Insert the capillary cartridge as described by manufacturer&#39;s protocol. If correctly installed, the inside light turns to &#34;blue&#34;.</li>
	<li>Remove the protective seal from the assay plate. Visually observe the pre-filled wells for air bubbles. If observed, pop them with a small pipet tip (Long-shaft P10 pipet tip works well).</li>
	<li>Load the plate holder by placing the assay plate and close the door. In computer, click on Start button.
	<ol>
		<li>Place ice tray containing temperature sensitive reagents and samples in the dark at 4 &#730;C until ready to prepare second pre-filled plate.</li>
		<li>Leave reagents/supplies out at room temperature for the second plate.</li>
	</ol>
	</li>
</ol>
5. Performing the CEI on plate 2
NOTE: This plate is set for analyzing phosphorylated TDP-43 levels.
<ol>
	<li>Remove the ice tray being stored at 4 &#730;C and place it on the bench, 1 h before the estimated completion time of the first plate. Retrieve a second plate and cartridge.</li>
	<li>Prepare any antibody dilutions needed for the second run and store them on ice. Prepare fresh 1:1 luminol-S:peroxide solution (step 3.12.8 above).</li>
	<li>Remix and briefly re-centrifuge the sample mix and reagents needed for loading the pre-filled plate. Load the second plate according to Figure 1. Load a-p(S409-410-2) TDP-43 antibody solution in row C wells.</li>
	<li>When the first run is complete, discard the first plate and cartridge. Remove the cartridge and place in a sharp&#39;s container for disposal. Keep the stickers from the plate and cartridge for reference purposes.</li>
	<li>Close the software file and reselect the same template. The software will remember the settings from the prior run. Make any changes to the annotation as needed (i.e., changing the primary antibody).</li>
	<li>Repeat steps 4.8&#8211;4.10. Put away all 12&#8211;230 kDa master kit separation module reagents and supplies</li>
	<li>Discard left over CEI sample-mix, antibody dilutions, 0.1x sample buffer and luminol-S:peroxide mixes in accordance with university regulations.</li>
</ol>
6. Data analysis
<ol>
	<li>Once the run is completed, ensure that the following quality checks are performed.
	<ol>
		<li>In software, select the Show Standards icon and Graph View tab. Check all 25 capillaries for the peak alignments to internal fluorescent marker sizes. Correct the misalignments by selecting Force Standard or right-clicking on the incorrect peak then selecting Not a Standard. Perform this check for each new capillary.</li>
		<li>Click on Samples and the Single View icon. Select capillary #1 (biotinylated ladder) in the experiment tab. Review the peak alignments to molecular weight markers. Click on the peak in Graph View and select Remove Peak, if an incorrect selection of the peak is done by the software. 
		NOTE: As an example, the 12&#8211;230 KDa biotinylated ladder will show sizing peaks at 12 kDa, 40 kDa, 66 kDa, 116 kDa, 180 kDa, and 230 kDa. The sizing of the sample peaks will be incorrect if this step is not performed and will generating inaccurate results.</li>
		<li>View the electrophoretic movie and note if any abnormal migration occurred during the run.</li>
		<li>Derive data (e.g., peaks table, including molecular weight, peak area, peak height, and signal-to-noise [S/N]) as needed for further calculations. There are graph annotation tools located at the upper-right corner of Graph window for providing more information about the graph.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis-based+immunoassays:+principles+and+quantitative+applications.">Capillary electrophoresis-based immunoassays: principles and quantitative applications.</a> Electrophoresis. 29 (16), 3279-3295 (2008).</li><li>Fourier, A., 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=Development+of+an+automated+capillary+nano-immunoassay-Simple+Western+assay-to+quantify+total+TDP43+protein+in+human+platelet+samples.">Development of an automated capillary nano-immunoassay-Simple Western assay-to quantify total TDP43 protein in human platelet samples.</a> Analytical and Bioanalytical Chemistry. 411 (1), 267-275 (2019).</li><li>. Compatibility, S.W.S.A.B Available from: <a target="_blank" href="https://www.proteinsimple.com/technical_library.html">https://www.proteinsimple.com/technical_library.html</a> (2017)</li></ol>

The authors declare no competing financial interest except ProteinSimple, Inc. covered the publication cost of this manuscript.

The capillary electrophoretic-based immunoassay is now the method of choice for high-throughput sample analysis and drug screenings25. Small sample volumes, well-optimized assay components, user-friendly assay platform and instrumentation, reagent expenditure, and low CV percentage are primary advantages26,27. Although there are several methods for separating proteins in different assay modalities, the antibody-based CEI described here can be adapted by small laboratories that are engaged in blood-based biomarker development. The CEI assay technology used here provides reliable, reproducible, and sensitive measurements for TDP-4328 and its phosphorylated derivative5.
The CEI system also provides a multiplexing choice of analyzing TDP-43 and its phosphorylated derivatives simultaneously and providing the direct quantification of the target protein, if purified or recombinant target protein is available. Full-length recombinant TDP-43 protein is commercially available; however, recombinant phosphorylated TDP-43 derivative is not. Since phosphorylated TDP-43 is not commercially available, a predictive phosphorylation value (PPV) was implemented to assess TDP-43 profile in ALS patients. Pan TDP-43 and phosphorylated TDP-43 amounts were permanently labelled with a fluorophore; therefore, the TDP-43 profile remains the same with or without a quantitative unit (i.e., ng/mL, pg/mL, etc.). Although determining the absolute amount of TDP-43 and its phosphorylated derivatives (i.e., P [S409-410-12] TDP-43) provides a more quantitative measurement, calculating PPV eliminates the necessity of the recombinant phosphorylated TDP-43 for standardization, since it is not commercially available.
CEI provides several checkpoints in the assay platform to accurately identify the problem in the case that an assay fails. This eliminates obstacles and provides better experimental design. The assay procedure is fully automated except for filling the sample plate. This is a significant feature compared to standard western blotting analysis. This feature provides consistency from run-to-run. Although every laboratory has unique standard operating procedures, it is important to adhere to practices that minimize human error. For example, it is critical to prepare the luminol-S/peroxide mixture just before plate loading, since adding peroxide into luminol starts the enzymatic reaction and consumes the luminol substrate. Loading samples and primary/secondary antibodies into plate wells without air bubbles are also critically important steps.
Additionally, since the plate wells are small in volume and there is no space between wells, users should use caution while pipetting, which is the most important step since everything else is automated. The loading order of the samples, antibodies, and other reagents is important for consistency of the assay (Figure 1). The process of plate preparation takes about 40&#8211;45 min. Therefore, it is recommended to first load the plate with the required assay components and prepare the luminol-S/peroxide mixture just before pipetting. This way, there is a consistent sequence of reagent adding, and consistent luminescence signal strength will be attained. It is not recommended to use an expired luminol-S/peroxide reagent, as it primarily affects the strength of the peroxide. Recent progress in introducing the split-buffer system and including the chemical and detergent compatibility range has enhanced the assay quality and produced more reproducible and predictable results. Now, a new combo-analyzer from the same manufacturer possesses a feature for analyzing the samples labeled with chemiluminescence and fluorescence conjugated antibodies in the same run. This new feature eliminates the need to consecutively run two individual plates and eliminates run-to-run variation.
The assay plates should be stored in ambient temperature. If it is chosen to keep the assay plates in a 4 &#730;C refrigerator, the plates must be taken out the night before the assay and brought to ambient temperature. Incorrectly loaded sample wells need to be extensively (4&#8211;5 times) washed with buffer provided in kit before adding the correct sample. Each primary antibody and biological samples are unique; therefore, the antibody/protein optimization should be performed before analyzing the samples for target proteins in biological fluids.
Here, the primary antibody incubation time was set for 30 min by default. If the signal is weak, users should consider increasing the primary antibody incubation time until reaching the desired signal strength without fluorescence signal burnout. For human platelets, pooled samples from patients were prepared and used for an optimization assay. The sample pooling better represents the variation among target biomolecules. It is recommended to use clear supernatants rather than total lysate or total homogenate for the CEI.
The high concentration of protein in whole platelet lysate mixture may decrease the signal-to-noise ratio (Figure 2). Repeated freeze-thaw cycles of samples should be avoided, as this adversely affects primary antibody binding. The ingredients of the lysate buffer are important, as some reagents are not compatible with CEI29. It is advised to cross-check the list of compatible reagents provided on the manufacturer&#39;s website before sample preparation. This is a limitation of the system that does not tolerate high stringency conditions for sample preparation. It is recommended to optimize the assay run parameters (i.e., primary antibody dilution, protein concentration, primary antibody incubation time, etc.) using pooled samples to subsequently analyze the individual samples.

The overall goal of CEI as described here is to provide an updated stepwise protocol for analyzing target proteins in human platelets. Assignment of a blood-based signature molecule is one of the most important tasks in the field of biomarker development in human neurodegenerative diseases, such as Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Parkinson&#39;s disease (PD), inclusion body myositis (IBM), and other protein-aggregation relevant pathologic conditions. The detection of minute amounts of such signature proteins in large volumes of blood with many interfering agents is a challenge. Therefore, the specificity, sensitivity, ability to handle large number of samples, and reproducibility of the selected method are crucial.
Human platelets can serve as a milieu to identify and assign potential biomarker proteins for neurodegenerative disease. Platelets provide the opportunity to serve as a surrogate primary cell model, which reflect some features of neuronal cells1,2,3. There are certain features that make platelets one of the preferred means to analyze biomarker candidate proteins and their chemical derivatives. First, platelets can be easily acquired using a less invasive approach by collecting blood from donors (i.e., venipuncture) or in large volumes from community blood banks. Second, platelets can be easily isolated from the whole blood with minimal preparatory work in minimally equipped laboratories4,5. Third, platelets do not have nuclei; therefore, they are a good model cell to study alterations in metabolism without transcriptional regulation. Fourth, the biomolecule content of platelets is encapsulated; therefore, the platelet microenvironment protects its contents from serum-interfering substances (i.e., proteases). Fifth, platelet-enriched plasma can be stored at room temperature for 7&#8211;8 days without losing metabolic activity. Therefore, platelets provide a working model in which external factors are minimized and controlled.
Traditional immunoassay techniques such as immunoblotting (e.g., western blotting) and enzyme-linked immunosorbent assay (ELISA) are more widely used in specific protein analysis. However, these two methods have several disadvantages, including multiple assay steps, requirement of hazardous chemicals and reagents, large sample size, issues with assay reproducibility, and inter-run data variabilities. These prompted the development of a method that is simpler with fewer steps and achievable in a relatively short period. Although the classical western blot technique will remain a popular laboratory method, its multi-step procedure, supplies, toxic waste (i.e., acrylamide, methanol, etc.) and assay time are becoming less desirable when performing high-throughput quantitative protein analysis.
An automated CEI approach is gradually becoming a method of choice for laboratories that conduct high-throughput protein assays6. CEI eliminates the need for gels, gel electrophoresis apparatuses, membranes, electrophoresis and electro-transfer devices, and more physical handling involvements. If designed well, a CEI assay should be completed within approximately 3.5 h, including quantitative data analysis, publication quality electropherogram, and graphs with statistical analysis. Another superiority of the CEI system is its requirement of 10x&#8211;20x less protein concentration, making it ideal for use in human samples used in clinical trials7,8.
The most critical part of CEI is optimizing the assay conditions for each antibody purchased from different vendors, type of antibody (monoclonal vs. polyclonal), optimum protein concentrations, sample preparation, sample denaturation temperature, and electrophoresis voltage applied on the capillaries. We have developed a single-assay format optimization method for the CEI that should be implemented before any new assays, which will save time and resources. This optimization step is followed by an automated quantitative assessment of both total and phosphorylated derivative of transactivation response DNA/RNA binding protein (TARDP). Due to its size (43 kDa), the acronym TDP-43 will be used throughout this paper. Here, TDP-43 protein in human platelet lysate obtained from ALS patients are assessed to help develop predictive phosphorylation value (PPV) as a potential prognostic biomarker.
TDP-43 is a new potential disease biomarker candidate for ALS. TDP-43 is an omnipresent protein in all nucleated cells; therefore, the functions of TDP-43 during various normal cellular events and in neurodegenerative disease have been investigated9,10,11,12,13,14. Although TDP-43 is a nuclear protein15, it has the ability to shuttle in and out between the nucleus and cytoplasm due to the presence of nuclear localization and nuclear export sequences16,17,18,19. Cytoplasmic TDP-43 is involved in various cellular events, such as mRNA stability and transport, the stress response, mitochondrial function, autophagosome20. However, not much is known about the role of phosphorylated derivatives of TDP-43 other than their involvement in the pathogenesis of neurodegenerative disease21.
This protocol illustrates how to optimize the assay conditions to analyze the contents of TDP-43 and its phosphorylated derivative in platelets using the CEI approach. Since phosphorylated TDP-43 is not commercially available, it is proposed to use a predictive phosphorylation value (PPV) to assess TDP-43 profiles in ALS patients. This CEI system utilizes a small volume of sample mixture (2.5&#8211;3.0 &#181;L per capillary). Total assay volume set-up is 8.0 &#181;L per capillary based on the manufacturer&#39;s protocol; hence, researchers can utilize one sample mixture preparation for two separate runs. The manufacturer designed the assay protocol so that any pipetting errors are minimized, if not entirely eliminated. The 24 individual human platelet lysate sample mixtures are divided into half-volumes (i.e., 2.5&#8211;3.0 &#181;L per sample) and consecutively analyzed those by two different antibodies within ~7 h. The CEI system described here provides a desirable high-throughput assay modality. Users need to test antibodies from different vendors and sample preparation modalities for the target protein before performing large-scale screening.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-230 kDa Separation kit</td>
			<td>ProteinSimple</td>
			<td>SM-W004</td>
			<td>Contains pre-filled assay plate and 25-channel capillary cartridge</td>
		</tr>
		<tr>
			<td>3000G Thermocycler</td>
			<td>Techne</td>
			<td>FTC3G/02</td>
			<td>We used this thermocylcer for heating the sample mix</td>
		</tr>
		<tr>
			<td>Anti-Mouse detection kit</td>
			<td>ProteinSimple</td>
			<td>042-205</td>
			<td>Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker</td>
		</tr>
		<tr>
			<td>Anti-P(S409-410) TDP-43 antibody</td>
			<td>ProteinTech</td>
			<td>22309-1-AP</td>
			<td>Primary antibody that recognizes phosphorylated TDP-43</td>
		</tr>
		<tr>
			<td>Anti-P(S409-412) TDP-43 antibody</td>
			<td>CosmoBio-USA</td>
			<td>TIP-PTD-P02</td>
			<td>Primary antibody that recognizes phosphorylated TDP-43</td>
		</tr>
		<tr>
			<td>Anti-Rabbit detection kit</td>
			<td>ProteinSimple</td>
			<td>DM-001</td>
			<td>Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker</td>
		</tr>
		<tr>
			<td>Anti-TDP-43 (pan) antibody</td>
			<td>ProteinTech</td>
			<td>10782-2-AP</td>
			<td>Primary antibody that recognizes whole TDP-43 protein</td>
		</tr>
		<tr>
			<td>Compass for SimpleWestern (SW)</td>
			<td>ProteinSimple</td>
			<td>Ver.4.0.0.</td>
			<td>Compass for SW is the control and data analysis application for SimpleWestern instruments</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator; Model100</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Sonicator. Used to rupture the cell membrane. This model is discontinued (Model XL2000-350)</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Eppendorf</td>
			<td>22625004</td>
			<td>Model# 5810 with swinging plate bucket</td>
		</tr>
		<tr>
			<td>Wes analyzer</td>
			<td>ProteinSimple</td>
			<td>55892-WS-2203</td>
			<td>Performs the capillary gel electrophoresis</td>
		</tr>
	</tbody>
</table>

use of capillary electrophoresis immunoassay to search for potential biomarkers of amyotrophic lateral sclerosis in human platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Optimization of platelet cytosolic protein concentration and primary antibody titration 
It is important to establish a linear dynamic range of platelet cytosolic proteins in the assay, since changes in signal are directly proportional to changes in protein in the platelet cytosol. Use of whole platelet lysate mixture in the assay may reduce the signal intensity of the target proteins (TDP-43 and P(S409-412) TDP-43) and contribute to a high background signal. Therefore, in this assay, the clear supernatant was used (cytosolic fraction) after rupturing platelets (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60638/60638fig02v2.jpg" /> 
Figure 2: Signal clarity depends on the sample quality. (A) Whole platelet lysate homogenate interferes with anti-TDP-43 antibody binding; therefore, a noisy electropherogram was observed. (B) Platelet cytosolic fraction was obtained from subjecting the whole lysate to centrifugation (16,000 x g for 30 min). Most of the membranous proteins were removed; hence, anti-TDP-43 antibody binding to TDP-43 protein was enhanced (blue line trace). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A linear dynamic range for platelet cytosol protein concentration was established at 0.2&#8211;0.8 mg/mL. An assay template was adopted so that both protein concentration and primary antibody titration were able to be performed in one assay (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60638/60638fig03v2.jpg" /> 
Figure 3: Linear dynamic range for platelet cytosol protein concentration. (A) Both protein concentrations and antibody titrations were optimized in one plate during the same run. (B) The linear working range (0.2&#8211;0.8 mg/mL) for protein was established. A 0.5 mg/mL protein load was labeled by a-ERK antibody as the positive control (capillary 25). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It should be noted that glycerol content in the sample preparation tube should be less than 20% (final), otherwise the high glycerol concentration will adversely affect the primary antibody binding.
Determining optimum exposure time 
In the older version of the software, the optimum exposure time had to be determined by plotting peak area against protein concentration (mg/mL). The new version provides a new tool named the high dynamic range (HDR) detection profile (Figure 4). Using the image panel provided the option to view all exposure times (i.e., 5 s, 15 s, 30 s, 60 s, 120 s, 240 s, 480 s) together. Computer software analyzed all exposure times and automatically identified the best exposure time (HDR). HDR detection profile delivered a significantly wider dynamic range due to the greater sensitivity of CEI, which means better detection and quantitation over a larger sample concentration range. However, users still have the option to choose any exposure time that satisfies the experimental goal. Using this feature, optimum exposure time was found for TDP-43 protein. The peak represents the optimum exposure time (Figure 4A). A single exposure time (4 s) was defined for this antibody after reviewing all nine exposure times ranging between 1&#8211;512 s (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60638/60638fig04v2.jpg" /> 
Figure 4: High dynamic range (HDR) detection profile for a target protein. (A) TDP-43 protein peak represents the optimum exposure time for the target protein. a-TDP-43 Ab titration was 1:300, and platelet cytosol protein concentration was 0.5 mg/mL. The software-defined blue line indicates the optimum exposure time. (B) This figure represents a user-defined single exposure time (4 s) after reviewing all nine exposure times ranging between 1&#8211;512 s. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
TDP-43 levels in human platelet cytosol of ALS patients 
A blood base biomarker development was performed. Using optimized assay conditions, platelet lysate cytosolic fractions obtained from ALS patients were analyzed using two sets of antibodies (i.e., anti-TDP-43 [Pan] antibody, an antibody that recognizes phosphorylated derivatives of TDP-43 protein; here, a-P [S409/410-2] TDP-43 was used). In this demonstration, disease-specific TDP-43 and its phosphorylated derivative are presented (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60638/60638fig5a.jpg" /> 
Figure 5: A representation of predictive phosphorylation value (PPV) of TDP-43. Absolute amount of phosphorylated TDP-43 and pan TDP-43 alone did not show much difference between the ALS and control groups. However, PPV indicated a slight increase in the ALS cohort, although there was no statistical difference between the two groups due to insufficient numbers of subjects (ALS = 25, control = 27). A low Cohen&#39;s d value between the means of ALS and control group showed a low effect size between the two groups due to small sample size (control = 25, ALS = 27). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Total TDP-43 was quantified using the calibration curve (Figure 6)
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60638/60638fig06v2.jpg" /> 
Figure 6: Standard calibration curve. Commercially purchased recombinant TDP-43 protein concentrations were used to construct a standard curve. Each data represents the average of triplicates. The protein band intensities were concentration dependent (Inset). <a href="https://www.jove.com/files/ftp_upload/60638/60638fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The quantification of phosphorylated TDP-43 protein was not possible due to commercially unavailability of this protein. Instead, a predicted phosphorylation value (PPV) was established that defines the percent of the phosphorylated species of TDP-43. PPV was determined from two sequential CEI assays for the same sample using the following equation.
<img alt="Equation 1" src="/files/ftp_upload/60638/60638equ01B.jpg" />
Intra- and inter-run assay variability were tested in pooled human ALS platelet cytosolic fractions (Figure 7)
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60638/60638fig07v2.jpg" /> 
Figure 7: Assay variations. (A) Two capillaries loaded with the same sample were analyzed by CEI, and intra-run assay variation was calculated as CV% = 14.9. (B) The same sample was analyzed on three different assay days and in three different assay runs. Inter-run assay variation was calculated as CV% = 18.7. <a href="https://www.jove.com/files/ftp_upload/60638/60638fig7alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although intra-run (capillary-to-capillary variation) coefficient variation values fell in the acceptable range (CV% = 14.9), the inter-run assay value was relatively high (CV% = 18.7). It is interpreted that this variation may be due to using capillary cartridges and sample plates from different lots. It is recommended that reproducibility studies should be performed in CEI components that have the same lot number.
Table 1: CEI assay plate loading template. <a href="https://cloudflare.jove.com/files/ftp_upload/60638/Table_1_Wes_Assay_Layout_single_sheet.xlsx" target="_blank">Please click here to download this table.</a>
Table 2: Interactive sample mixture preparation template. After entering the stock protein concentration from unknown samples, interactive cells will automatically calculate how much volume needs to be used for preparing the sample mix. <a href="https://cloudflare.jove.com/files/ftp_upload/60638/Table_2_Wes_Assay_Sample_mix_preparation_template_UD.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets at JoVE.com

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

use-capillary-electrophoresis-immunoassay-to-search-for-potential

Research

JoVE Journal

Neuroscience

7.1K Views.  Kansas City University of Medicine and Biosciences. Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Video: Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method's advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.

Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli.

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Event Related Potentials (ERPs) and other EEG Based Methods for Extracting Biomarkers of Brain Dysfunction: Examples from Pediatric Attention Deficit/Hyperactivity Disorder (ADHD)

Neuropsychiatric diagnoses like ADHD are based on subjective methods like interviews, rating scales and observations. There is a need for more brain-based supplements. Stimulant medication is the most common treatment for ADHD. Clinically useful predictors of response have so far not been reported. The aim of this paper is to describe the EEG based methods we apply to extract potential biomarkers for brain dysfunction. Examples relate to biomarkers for pediatric ADHD, and prediction of medication response. The main emphasis is on Event Related Potentials (ERPs).
A nineteen channel EEG is recorded during a 3 min eyes-opened task, a 3 min eyes-closed task, and a 20 min cued visual GO/NOGO task (VCPT). ERPs are recorded during this task. The goal of the ERP protocol is to extract biomarkers of assumed brain dysfunctions that significantly differentiate between a patient group and healthy controls. The protocol includes recording during standard conditions and artifact correction. ERP waves can be used or transformed into latent components. The components of the patient group are compared with controls, empathizing components that, when compared, show relatively high effect sizes. Sub-groups of the patients are selected on the basis of the cluster analysis in the space of the components. Treatment procedure (such as medication, tDCS or neurofeedback protocol) can be applied and the changes in components related to treatment in the subgroups are observed, forming the basis for clinical recommendations.
The methods described were applied in a study of 87 pediatric ADHD patients. The index of medication response discriminated significantly between responders and non-responders with a large, and clinically meaningful effect size (d = 1.84). In an ongoing study comparing ADHD children with matched controls, several variables discriminate significantly between patients and controls. The global index will exceed d = .8. The EEG based methods described here could be clinically meaningful.

Event Related Potentials ERPs and other EEG Based Methods for Extracting Biomarkers of Brain Dysfunction: Examples from Pediatric Attention Deficit/Hyperactivity Disorder ADHD

EEG-methods are applied for extracting biomarkers of brain dysfunctions. The focus is on multi-channel event-related potentials (ERPs) recorded in a cued GO/NOGO task. Non-brain artifacts are corrected and ERPs are compared with the normative data. Examples relate to biomarkers for ADHD diagnosis and prediction of medication response.

Neuropsychiatric diagnoses like ADHD are based on subjective methods like interviews, rating scales and observations. There is a need for more brain-based ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...