This work was supported by de.NBI, a project of the German Federal Ministry of Education and Research (BMBF) (grant number FKZ 031 A 534A) and P.U.R.E. (Protein Research Unit Ruhr within Europe) and Center for Protein Diagnostics (ProDi) grants, both from the Ministry of Innovation, Science and Research of North-Rhine Westphalia, Germany.

The use of human brain tissue was approved by the ethics committee of the Ruhr-University Bochum, Germany (file number 4760-13), according to German regulations and guidelines. This protocol has been applied on commercially obtained substantia nigra pars compacta tissue slices. A graphical overview of the presented protocol is shown in Figure 1.
1. Tissue sectioning
<ol>
	<li>Precool the cryostat chamber. 
	NOTE: Every tissue requires different cryostat temperatures, which can be found in the respective vendor protocol.</li>
	<li>Clean the stainless-steel knife with 70% ethanol and install it into the blade holder.</li>
	<li>Transfer the tissue from the -80 &#176;C freezer to the cryostat using an icebox and let it adjust to the cryostat chamber temperature for 15 min.</li>
	<li>Unambiguously label membrane slides using a pencil. 
	NOTE: PET/PEN membrane slides are required for the LMD-based sample collection. Handle the PET/PEN membrane slides with care as they are extremely fragile.</li>
	<li>Apply a drop of commercial frozen section medium on the tissue holder. Before it is completely frozen, place the tissue onto the frozen section medium and let it harden, so that the tissue is connected with the tissue holder.</li>
	<li>Install the tissue holder in the cryostat chamber and adjust its orientation before you start sectioning. Optimal holder orientation depends on the orientation of the tissue. 
	NOTE: It may be necessary to trim the tissue until the section plane needed for the slices is reached.</li>
	<li>Before the tissue area of interest is reached, adjust the cutting setting to the desired tissue thickness. 
	NOTE: 5 or 10 &#181;m is the suggested thickness for this protocol as 20 &#181;m thick sections were found to be incompatible with the LMD-based sample collection22.</li>
	<li>Cut two sections and discard them.</li>
	<li>Put down the Anti-roll Plate.</li>
	<li>Cut a section of the tissue, open the Anti-roll Plate carefully, take a membrane slide, and prevent tissue folding while placing the tissue section on the membrane slide. 
	NOTE: Storing the membrane slides at room temperature prior to adhesion enables accurate sample attachment. Several sections may be placed on the same membrane slide but tissue overlapping must be prevented.</li>
	<li>Store tissue sections placed on membrane slides in the cryostat until sectioning is completed.</li>
	<li>Store the cryosected tissue at -20 &#176;C until further processing or directly proceed with the procedure below. Store the sectioned tissue slides at -80 &#176;C until further use.</li>
</ol>
2. Laser Microdissection and Pressure Catapulting
NOTE: As neuromelanin granules are visible without any staining due to their black-brownish color, no staining is necessary for this protocol. Nevertheless, different staining procedures can be combined with this protocol if required. Keep in mind that the use of blocking solutions or antibodies will influence the LC-MS/MS analyses.
<ol>
	<li>Switch on the MicroBeam system and open the associated software on the computer (see Table of Materials).</li>
	<li>Place the tissue membrane slide in the SlideHolder on the RoboStage with the tissue facing upwards. 
	NOTE: Depending on the LMD device, it may be necessary to place the membrane slide such that the tissue is facing down. In general, sample collection is performed in a temperature-controlled environment to ensure optimal and reproducible conditions.</li>
	<li>Set the microscope to the desired magnification (50-fold is used here) for the overview scans.</li>
	<li>Use the Scan function, which can be found in the Navigator window of the software interface, to acquire an overview scan of the tissue section. Search for the top-left corner and the bottom-right corner of the area of interest and select them in the software interface. Then, select Scan all ROIs to perform the scans. 
	NOTE: Overview scans are not mandatory, but they enable better orientation in the slide and can be saved for later usage.</li>
	<li>Adjust the magnification of the microscope for the appropriate tissue, which is 400-fold in the present case of neuromelanin granules.</li>
	<li>Search for an area with neuromelanin granules. Select Field of View Analysis in the software interface, select Invert Result, and set the threshold for the RGB channels so that only neuromelanin granules are highlighted in red in the preview window. Click on OK to use the adjusted settings for the field of view. 
	NOTE: It may occur that smaller objects having a dark color also get selected. To account for that, discard all objects covering an area smaller than 100 &#181;m2 before isolating neuromelanin granules. To do this, open the Element List by clicking on the icon in the toolbar, select the slide under consideration and order elements by area. Select those with areas smaller than 100 &#181;m2 and delete them.</li>
	<li>Adjust laser settings using an area of the slide that is covered by the membrane only. 
	NOTE: It is suggested to use the Cut Laser Adjustment Wizard and follow the instructions of the software. Required laser settings may differ between different slides. For 5 &#181;m sections with 400-fold magnification, typical settings are 32 energy and 51 focus for cutting, and 28 energy and -1 focus for laser pulse catapulting (LPC).</li>
	<li>Adjust speed settings for positioning and cutting to ensure proper isolation. 
	NOTE: 30% speed was found to be optimal for NMG isolation.</li>
	<li>Fill the sample collection tube cap with 50 &#181;L ultrapure water and insert the cap into the collector of the RoboMover. 
	NOTE: The tube collector used for present experiments can carry one sample collection tube at a time.</li>
	<li>Position the RoboMover above the RoboStage II using the software interface to start sample collection. 
	NOTE: To do this, open the RoboMover window, which displays the collector. Click on the sample collection tube cap displayed in the RoboMover window to move the cap to the working area. Adjust the optimal moving and working height in the RoboMover window. Otherwise, the water in the cap may drop onto the slide or the catapulted objects will not reach the cap.</li>
	<li>Start the laser. Control energy and focus settings during the laser process and adjust the settings if necessary. Ensure proper isolation and catapulting of the isolated objects into the sample collection tube cap for at least the first ten objects. 
	NOTE: Proper isolation and catapulting have to be checked visually. Both should result in a tissue-free area of the size of the pre-selected object in the tissue slice (see Figure 2C,D). Adjust the laser settings if the object stays attached to the tissue slice after cutting and catapulting. For catapulting, the CenterRoboLPC option is found to be well suited for NMG isolation. The catapulting settings can be adjusted for each selected object in the Element List.</li>
	<li>When sampling is completed, navigate the RoboMover to its starting position. Remove the sample collection tube. 
	NOTE: When the number of collected objects is rather low and objects are big enough, sample collection can be ensured by clicking on Cap Check, which will place the sample collection tube cap under the microscope so that the number of objects inside of the water in the cap can be counted (see Figure 2H).</li>
	<li>Spin down the sample using a centrifuge. Short spins of 5 s with increasing centrifugal force due to acceleration of the centrifuge were found to be sufficient. At this point, store samples at -80 &#176;C, as all samples should be further processed together. 
	NOTE: For the comparison of the proteomic profile, the tissue surrounding the NMGs was also isolated after their excision. The isolation of the surrounding tissue was performed at 50-fold magnification.</li>
	<li>Dry the samples in a vacuum concentrator. 1.5 h were found to be sufficient for 50 &#181;L of water.</li>
	<li>Solubilize and lyse the tissue in 40 &#181;L formic acid for 20 min (room temperature).</li>
	<li>Enhance tissue lysis by sonication at 45 kHz (kilohertz) for 5 min in a sonication bath. Fill the sonication bath with ice to prevent tubes from melting. Store the samples at -80 &#176;C until further processing.</li>
</ol>
3. Tryptic digestion
<ol>
	<li>Defreeze samples on ice.</li>
	<li>Completely dry the samples in a vacuum concentrator.</li>
	<li>Fill up the sample with 50 &#181;L of a suitable digestion buffer, e.g., 50 mM ammonium bicarbonate.</li>
	<li>After the addition of 1.25 &#181;L of 200 mM 1,4-dithiothreitol, incubate the samples for 30 min at 60 &#176;C and 300 rpm using a thermomixer and cool them down to room temperature (RT) afterwards.</li>
	<li>Then, incubate samples at RT for 30 min in the dark after the addition of 1.36 &#181;L 0.55 M iodoacetamide.</li>
	<li>Add a suitable amount of trypsin to the samples and incubate the samples overnight (~16 h) at 37 &#176;C. 
	NOTE: For 1,000,000 &#181;m2, 0.1 &#181;g of trypsin was found to be sufficient.</li>
	<li>Add 2.6 &#181;L of 10% trifluoroacetic acid (TFA) to the samples to stop the digestion (end concentration of 0.5% TFA).</li>
	<li>Completely dry the samples using a vacuum concentrator. Then, fill samples up to a defined final volume with 0.1% TFA. NMG samples were filled up to 20 &#181;L of which 5 &#181;L were used for one mass spectrometric (MS) experiment.</li>
	<li>Store the samples at -80 &#176;C until further usage. Determine peptide concentration by amino acid analysis or another suitable quantification method (e.g., Direct Detect). 
	&#8203;NOTE: Low sample amounts may not be quantifiable using the mentioned techniques. To ensure identical sample loading, each sample should contain the same amount of isolated tissue and every sample should be treated equally.</li>
</ol>
4. High-performance liquid chromatography and mass spectrometry
NOTE: The following high-performance liquid chromatography (HPLC) mass spectrometric (MS) analysis are optimized for the specific LC system with a trapping column device and mass spectrometer used here (see Table of Materials). For other LC and MS systems, adaption of parameters is recommended.
<ol>
	<li>Using the software Xcalibur, adjust the HPLC settings as follows.
	<ol>
		<li>Trap column: Set temperature to 60 &#176;C, flow rate to 30 &#181;L/min, running buffer to 0.1% trifluoroacetic acid.</li>
		<li>Analytical C18 reversed-phase column: Set temperature to 60 &#176;C, flow rate to 30 &#181;L/min, running buffer A to 0.1% trifluoroacetic acid, running buffer B to 84% acetonitrile, and gradient to 5%-30% running buffer B over 98 min. 
		NOTE: Adaption of the gradient may be inevitable and is strongly recommended when using different tissues or cells. Total gradient time may vary due to sample loading at the beginning of the gradient and sample washing at the end of the gradient. The total gradient in this protocol consists of 7 min sample loading and additional column wash for 15 min resulting in a total gradient time of 120 min.</li>
	</ol>
	</li>
	<li>Create a data-dependent acquisition (DDA) method using the XCalibur Instrument Setup, which can be found in the HPLC software roadmap menu.</li>
	<li>In the Global Parameters tab, define the infusion mode Liquid Chromatography, the Expected LC Peak Width (30 s), and the Default charge state (2).</li>
	<li>Proceed to the Scan Parameters tab and add the following scans and filters in the order mentioned: MS OT, MIPS, Intensity, Charge State, Dynamic exclusion, and ddMS2 OT HCD. 
	NOTE: The detailed parameter settings for each scan and filter can be found in Supplementary Table 1. Optimal MS and DDA settings might vary for the specific mass spectrometer used as well as the sample type and should be, therefore, adapted.</li>
	<li>Prepare samples by dissolving 200-400 ng of sample peptides in a defined volume of 0.1% TFA in inert mass spectrometric glass vial inlets. If concentration determination is not applicable due to low sample amount, verify identical sample loading by comparing the Total Ion Current (TIC). 
	NOTE: To do this, open the resulting file of mass spectrometric measurement in a suitable software, e.g., FreeStyle, and check the chromatogram. Intensities should be comparable for all samples. A representative TIC is shown in Figure 3.</li>
	<li>Analyze the raw data obtained using a proteomic suitable software, e.g., MaxQuant23, Progenesis QI for Proteomics, or Proteome Discoverer, and perform a statistical data analysis based on the research question.</li>
</ol>
5. Analysis of proteomic raw data using MaxQuant
NOTE: A detailed information on MaxQuant parameters is provided in Supplementary Table 2. They are briefly described below.
<ol>
	<li>Load raw files into the MaxQuant software in the raw data header by clicking Load.</li>
	<li>Assign sample names by clicking on Set Experiment.</li>
	<li>Define group-specific parameters. First, add modifications. Due to sample processing, choose Deamidation (NQ), Oxidation (M), and Carbamidomethylation (N-term) as variable modifications, and add Carbamidomethylation (C) as fixed modification.</li>
	<li>Choose Trypsin as digestion enzyme in the Digestion tab.</li>
	<li>Add the label free quantification option LFQ in the Label Free Quantification tab. If more than 10 files are to be processed, choose the Fast LFQ option to shorten the processing time. Add iBAQ option as a measure for protein quantification24.</li>
	<li>Ensure that all other group-specific parameters remain in factory settings.</li>
	<li>Proceed to the Global Parameters tab and add the FASTA file derived from uniprot.org in the Sequences tab. Modify the identifier rule accordingly and add the taxonomy ID, in this case, 9606 for homo sapiens.</li>
	<li>For protein quantification choose Unique and Razor peptides.</li>
	<li>Ensure that all other global parameters remain in factory settings.</li>
	<li>Click on Start and retrieve the proteingroups.txt output after MaxQuant analysis for further analysis in Perseus.</li>
</ol>
6. Statistical analysis using Perseus
<ol>
	<li>Load the proteingroups.txt file in Perseus, add the iBAQ values as main columns, and sort all other columns according to their type.</li>
	<li>Filter out decoys and contaminants by filtering rows based on the categorical column.</li>
	<li>Filter results based on valid values. In the present case, with only two samples included in the analysis, a minimum number of one valid value was chosen.</li>
	<li>Export the Perseus output in .txt format for further processing, for example, in Excel, and evaluate the results regarding the research question.</li>
</ol>
7. Validation of selected proteins
NOTE: Commonly used methods for validation of MS data are, for example, immunological staining or Western Blot. Due to the dark color and the autofluorescence of neuromelanin, immunological staining of proteins inside of neuromelanin granules either with horseradish peroxidase- or fluorophore-conjugated antibodies are not applicable. For Western Blot analysis, very large amounts of post-mortem tissue would be necessary. Therefore, selected proteins are validated by targeted mass spectrometry, and in the present case, parallel reaction monitoring (PRM)-experiments were set up.
<ol>
	<li>Select proteins for validation. Choose peptides of these proteins already detected in DDA experiments. Peptides should contain no missed cleavages or modifications to ensure a reliable quantification. 
	NOTE: There can be several reasons for the validation of one specific protein, for example, differential abundances in the investigated conditions. For the representative results, cytoplasmic dynein 1 heavy chain 1 has been selected, which was found to be equivalently abundant in NMG and SN samples and could therefore be used as a reference to ensure equal sample loading.</li>
	<li>Use the selected peptides to set up the first version of a PRM-method using the HPLC software. Keep all chromatography and global parameters settings from the DDA method.</li>
	<li>Add MS OT and tMS2 OT HCD as scan types. Ensure that the settings for MS OT are the same as for the DDA method. Detailed settings for the PRM method can be found in Supplementary Table 1.</li>
	<li>For tMS2 OT HCD, add selected peptides as an inclusion list. Therefore, add the amino acid sequence and the m/z value observed in the DDA measurements. For the first PRM experiment, do not add retention time windows or set t start to 0 and t stop to 120 (for a 120 min gradient).</li>
	<li>Evaluate the PRM method after the measurement using suitable software, for example, Skyline, and obtain the retention time of the peptides added to the inclusion list. For included peptides, check that comparable peaks are observable for at least three precursor ions in MS1 scans and five fragment ions in MS2 scans with low mass error (&#177;5 ppm).</li>
	<li>Refine the PRM method, for example, by increasing the resolution for the tMS2 OT HCD scan and adding retention time windows to the inclusion list. 
	NOTE: Retention time windows of 3 min were found to be well-suited in present experiments (observed retention time in first PRM experiment &#177; 1.5 min).</li>
	<li>With the refined PRM-method, perform quantification of peptides and proteins of interest based on the peak area both on MS1 and MS2 levels with suitable software.</li>
</ol>

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Venny: An interactive tool for comparing lists with Venn's diagrams Available from: <a target="_blank" href="https://bioinfogp.cnb.csic.es/tools/venny/index.html">https://bioinfogp.cnb.csic.es/tools/venny/index.html</a> (2007)</li><li>Plum, S., 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=Combined+enrichment+of+neuromelanin+granules+and+synaptosomes+from+human+substantia+nigra+pars+compacta+tissue+for+proteomic+analysis.">Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis.</a> Journal of Proteomics. 94, 202-206 (2013).</li></ol>

The authors declare no conflicts of interest.

LMD is a widely applicable technique for the isolation of specific tissue areas, single cells, or subcellular structures. In the revised and automated protocol presented here, this technique is applied for the specific isolation of neuromelanin granules (NMGs) and NMG-surrounding tissue (SN). Until now, two different approaches for the isolation of NMGs out of human post-mortem brain tissue were published and widely used:
a) A discontinuous sucrose gradient consuming 1 g of substa...

Every tissue consists of a heterogeneous mixture of different cell types, but the specific isolation of one cell type often is indispensable for a more precise characterization. Laser microdissection (LMD), coupling a microscope with a laser application, is a powerful tool for the specific isolation of tissue areas, single cells, or cellular substructures out of a complex composite. The application of LMD in combination with mass spectrometry (LMD-MS) has already been successfully implemented for several research questions, including isolation of DNA1, RNA2 and proteins3,4,5. In this protocol, a revised and optimized LMD-MS protocol is described for the proteomic analysis of human post-mortem brain tissue and sub-cellular components to decipher novel pathomechanisms of Parkinson&#39;s disease.
Neuromelanin is a black, nearly-insoluble pigment found in the catecholaminergic, dopamine-producing neurons of the substantia nigra pars compacta6. Together with proteins and lipids, it accumulates in organelle-like granules surrounded by a double membrane, called neuromelanin granules (NMGs)7,8,9. NMGs can be observed from the age of three years in humans increasing in quantity and density during the aging process10,11. To date, there is no definite hypothesis on neuromelanin formation, but one assumption is that neuromelanin is formed through the oxidation of dopamine12. Other hypotheses are based on enzymatic production of neuromelanin (e.g., tyrosinase)13. Neuromelanin itself was found to have a high binding affinity to lipids, toxins, metal ions, and pesticides. Based on these findings, the formation of NMGs is assumed to protect the cell from the accumulation of toxic and oxidative substances and from environmental toxins14,15. Besides this neuroprotective function, there is evidence that neuromelanin may cause neurodegenerative effects, e.g., by iron saturation and the subsequent catalysis of free radicals16,17. Furthermore, neuromelanin released during neurodegenerative processes can be decomposed by hydrogen peroxide, which could accelerate necrosis by reactive metals and other toxic compounds previously bound to neuromelanin and may contribute to neuroinflammation and cellular damage18. However, until now the exact role of NMGs in neurodegenerative processes like in the course of Parkinson&#39;s disease is not clearly understood. Still, NMGs seem to be involved in the pathogenesis of Parkinson&#39;s disease and their specific analysis is of utmost importance to unravel their role in neurodegeneration. Unfortunately, common laboratory animals (e.g., mice and rats) and cell lines lack NMGs19. Therefore, researchers especially rely on post-mortem brain tissue for their analysis. In the past, NMG isolation by density gradient centrifugation relied on the availability of high amounts of substantia nigra tissue20,21. Today, LMD presents a versatile tool to specifically isolate NMGs from human brain samples to then analyze them by LC-MS/MS.
In this protocol, an improved and automated version of a previous protocol22 is presented for the isolation of NMGs and surrounding tissue (SN), enabling a faster sample generation, higher numbers of identified and quantified proteins, and a severe reduction of required tissue amounts.

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			<td>1,4-dithiothreitol</td>
			<td>AppliChem</td>
			<td>A1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Merck</td>
			<td>1.00029.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A6141</td>
			<td></td>
		</tr>
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			<td>Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>56302</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>AppliChem</td>
			<td>A1666,0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tube 500</td>
			<td>Carl Zeiss</td>
			<td>415190-9221-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGAAPFADBMBHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>PALM MicroBeam</td>
			<td>Zeiss</td>
			<td>494800-0014-000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN Membrane slide</td>
			<td>Carl Zeiss</td>
			<td>415190-9041-000</td>
			<td></td>
		</tr>
		<tr>
			<td>substantia nigra pars compacta tissue slices</td>
			<td>Navarrabiomed Biobank (Pamplona, Spain)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Merck</td>
			<td>91707</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin sequencing grade</td>
			<td>Serva</td>
			<td>37283.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimate 3000 RSLC nano LC system</td>
			<td>Thermo Fisher Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Software</td>
			<td>Weblink/Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeStyle</td>
			<td>Thermo Fisher Scientific</td>
			<td>1.6</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://www.maxquant.org/</td>
			<td>1.6.17.0</td>
			<td></td>
		</tr>
		<tr>
			<td>PALMRobo</td>
			<td>Zeiss</td>
			<td>4.6 pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Perseus</td>
			<td>https://www.maxquant.org/perseus/</td>
			<td>1.6.15.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Skyline</td>
			<td>https://skyline.ms/project/home/software/Skyline/begin.view</td>
			<td>20.2.0.343</td>
			<td></td>
		</tr>
		<tr>
			<td>XCalibur</td>
			<td>Thermo Fisher Scientific</td>
			<td>4.3</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser microdissection-based protocol for the lc-ms/ms analysis of the proteomic profile of neuromelanin granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

The specific isolation of NMGs and SN tissue is the most important step for the successful application of this protocol. Using the Field of View Analysis function in the vendor-provided software of the LMD, NMGs can be automatically selected in a color-dependent manner. Therefore, tissue areas containing NMGs (Figure 2A) have to be identified and a Field of View Analysis with adjusted color thresholds has to be performed, resulting in the labeling of NMGs (<strong class="xfi...

Watch this Scientific Journal Video about Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules at JoVE.com

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

laser-microdissection-based-protocol-for-lc-msms-analysis-proteomic

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Neuroscience

2.1K Views.  Ruhr-University Bochum. A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Video: Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...