studying alpha-synuclein accumulation in primary embryonic mouse dopamine neurons

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Add a 3.75 microliters of 100 microgram per milliliter of pre-formed fibrils to each experimental well, and 3.75 microliters of PBS to each control well.
When working with PFFs, be cautious about the unwanted protein contamination and afterwards, clean the hood and all PFF-associated instruments with 1% SDS and 70% ethanol.
At the appropriate experimental endpoint, after staining for the markers of interest, load the 96 well culture plate onto a high content plate scanner, fitted with a 10x objective. Adjust the settings according to the specifications of the 96 well plate, such as the plate type, manufacturer, size, distance between the wells, and the type and volume of medium.
Select the imaging area of the wells to cover all of the cells in each micro island, and use one well to adjust the autofocus on the DAPI expression. Calibrate the acquisition time for each fluorescent channel, based on the intensity of the staining in the control wells, and adjust the parameters so that the dopamine cells in the preformed fibril treated control wells, harboring phosphoserine 129 alpha synuclein aggregates within the cell soma, clearly be distinguished, allowing an unambiguous quantification of the phosphoserine alpha synuclein positive and negative cells.
Then, image all of the selected wells simultaneously in all of the channels, using exactly the same parameters for each well.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation
<ol>
	<li>Prepare dopamine neuron medium (DPM) with 0.46% D-glucose, 1% L-glutamine, 1% N2, 0.2% primocin, completed with DMEM/F12. Filter the DPM after mixing the ingredients. Store DPM at 4 &#176;C and warm each aliquot only once. 
	NOTE: DPM should not contain glia-derived neurotrophic factor&#160;(GDNF), as it will reduce &#945;-synuclein accumulation in dopamine neurons.</li>
	<li>Prepare siliconized glass pipettes that are extremely hydrophobic, thereby minimizing the attachment to the surface and loss of cells during the initial handling of embryonic neurons.
	<ol>
		<li>Add 10 mL of siliconizing fluid to 1 L of distilled water and mix by stirring in a 2 L vessel. Leave the glass pipettes immersed in the siliconizing solution for 15 min.</li>
		<li>Rinse the pipettes 3&#8211;5x with distilled water. Dry the pipettes overnight at room temperature (RT) or for 1&#8211;2 h at 100-120 &#176;C heated sterile space to speed up the drying.</li>
		<li>Sterilize the pipettes by standard autoclaving in a sealed autoclave bag.</li>
	</ol>
	</li>
	<li>Prepare poly-L-ornithine (PO) coated 96 well plates with transparent bottoms by adding 60 &#956;L of PO solution into the middle wells of the 96 well plate to be used for seeding of the neurons, leaving at least one row/column of wells at the edges of the plate to avoid edge effects. Keep the coated plate overnight at 4 &#176;C or 4 h at RT.</li>
	<li>Prior to plating the cells, aspirate PO completely and wash the cells thrice with 100 &#956;L of 1x Phosphate Buffer Saline (PBS). Aspirate 1x PBS from the wells and keep the lid of the plate open for complete drying. 
	NOTE: It is possible to collect used PO and filter it for reuse. This can be repeated twice for the same PO solution.</li>
	<li>Add 50 &#956;L of DPM to previously coated wells. Aspirate DPM from the wells with a 100 &#956;L plastic tip and simultaneously scratch the bottom of the well with circular movements to remove the coating at the perimeter of each well. A PO-coated island will remain in the middle of the well.</li>
	<li>Under a laminar hood, add 10 &#956;L of DPM to the middle of each coated island to create micro islands. 
	NOTE: A plate with DPM-covered micro islands can be kept under the laminar flow hood for 1&#8211;2 h during the isolation of cells.</li>
</ol>
2.&#160;Isolation of the ventral midbrain floor from E13.5 mouse embryos
NOTE: Refer to&#160;Figure 1&#160;for midbrain floor dissection steps.
<ol>
	<li>Prior to dissection, fill a 10 cm Petri dish with Dulbecco&#39;s buffer and keep it on ice.</li>
	<li>Euthanize an E13.5 pregnant female mouse according to the institution&#39;s guidelines. Place the mouse flat on its back and spray the anterior body with 70% ethanol. Lift the skin above the womb with forceps and make an incision with surgical scissors to expose the uterus.</li>
	<li>Carefully remove the uterus and place it into the previously prepared Petri dish on ice.</li>
	<li>Using surgical scissors under the laminar hood at RT, carefully remove the embryos from the uterus. Remove all placental residue from the embryos with forceps and place them into a new 10 cm Petri dish filled with Dulbecco&#39;s buffer.</li>
	<li>Using dissection forceps or needles, cut off the hindquarter of the head from the places marked with black arrows in&#160;Figure 1A. Take the cut piece away from the rest of the embryo (Figure 1B).</li>
	<li>Place the posterior of the cut piece towards the observer (Figure 1C) and gently cut it open from caudal to cranial (Figure 1D). From 0.5 mm below the cranial opening, cut a 2 mm2&#8211;3 mm2&#160;region, shown in&#160;Figure 1E.</li>
	<li>Collect the ventral midbrain floor (see&#160;Figure 1F) in an empty 1.5 mL microcentrifuge tube. Keep the microcentrifuge tube on ice until all midbrain floors are collected in it. 
	NOTE: Alternatively, the midbrain floors can be collected with a 1 mL micropipette after dissection of all embryo brains.</li>
</ol>
3.&#160;Establishing primary embryonic midbrain cultures from E13.5 mouse embryos in 96 well plate format
<ol>
	<li>After the collection of midbrain floors from all embryos in the same 1.5 mL tube, remove the residual Dulbecco&#39;s buffer and wash the tissue pieces thrice with 500 &#956;L of Ca2+, Mg2+-free Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Remove HBSS and add 500 &#956;L of 0.5% trypsin to the tube. Incubate it at 37 &#176;C for 30 min.</li>
	<li>During incubation, warm 1.5 mL of fetal bovine serum (FBS) at 37 &#176;C, add 30 &#956;L of DNase I to the FBS and mix. Also, fire-polish the tip of a siliconized glass pipette. Make sure that the hole has no sharp edges and is around the same size as a 1 mL micropipette tip. 
	NOTE:&#160;As an alternative, a low adhesion 1 mL micropipette tip can be used for trituration. However, siliconized glass pipettes seem to give the best results.</li>
	<li>As soon as the incubation in step 3.2 ends, add 500 &#956;L of the FBS/DNase mix to the partially digested tissue. Use the glass pipette to triturate the tissue in the FBS/trypsin mix. Triturate until tissues dissociate into tiny, barely visible particles. Avoid bubbles during trituration.</li>
	<li>Let the leftover particles precipitate at the bottom of the microcentrifuge tube by gravity. Without pipetting the precipitate at the bottom, collect the supernatant into an empty 15 mL conical polypropylene tube.</li>
	<li>Dilute FBS/DNase I from step 3.3 (98:2) with 1,000 &#956;L of HBSS to obtain FBS/DNase-I/HBSS (49:1:50). Mix by pipetting up and down. Add 1,000 &#956;L of the new mix to the leftover particles in the microcentrifuge tube. Triturate again and repeat step 3.5.</li>
	<li>Repeat the previous step once more to use up all FBS/DNase-I/HBSS (49:1:50).</li>
	<li>Once all the supernatant is collected inside the 15 mL tube (from steps 3.5, 3.7, and 3.8), use a tabletop centrifuge to spin down the supernatant (~3 mL) at 100 x&#160;g, for 5 min. Remove the supernatant without touching the pelleted cells at the bottom.</li>
	<li>Wash the cell pellet by adding 2 mL of DPM to the tube and spin it down at 100 x&#160;g&#160;for 5 min. Remove the supernatant and repeat the washing 2x to minimize the debris in the pelleted cells. 
	NOTE:&#160;Always use fresh, warmed DPM for the cultured neurons. For the washing steps, DPM does not have to be fresh but should be prewarmed to 37 &#176;C.</li>
	<li>Dilute the cells with fresh, warm DPM and transfer them to a microcentrifuge tube. The amount of DPM for dilution depends on the number of embryos used for tissue dissection. For example, use 150 &#956;L of DPM to dilute the cells obtained from ten embryos.</li>
	<li>Transfer 10 &#956;L of cells in DPM to a microcentrifuge tube. Mix them with 10 &#956;L of 0.4% Trypan blue stain. Count live (i.e., Trypan blue negative) cells using a hemocytometer or an automated cell counter. 
	NOTE:&#160;Use 30,000 cells for plating per well to obtain ~1,000 dopamine neurons per well. If the cell density is higher than ~30,000 cells per 6 &#956;L, further dilute the cells with DPM before plating so that the seeding volume is no less than 6 &#956;L.</li>
	<li>Without touching the bottom of the wells, remove the DPM from the micro islands created at step 1.6.</li>
	<li>In order to obtain reproducible cell density at each well, mix the cells by gentle pipetting prior to plating in the well. With a 1&#8211;10 &#956;L micropipette, add 6 &#956;L of cells to the middle of the well at the location of each former micro island.</li>
	<li>Fill the empty wells at the edges of the plate with 150 &#956;L of water or 1x PBS to minimize evaporation from the wells containing neuronal cultures. Incubate the plate in an incubator at 37 &#176;C, 5% CO2&#160;for 1 h.</li>
	<li>After 1 h, remove the plate from the incubator, add 100 &#956;L of DPM into each well with cells, and place it back in the incubator.</li>
	<li>Two days after plating (day in vitro 2, or DIV2), remove 25 &#956;L and add 75 &#956;L of fresh DPM to bring the final media volume to 150 &#956;L and avoid evaporation as much as possible.</li>
	<li>Exchange half of the medium with fresh DPM (i.e., remove 75 &#956;L and add 75 &#956;L fresh DPM) at DIV5. Do not perform any media changes after DIV5.</li>
</ol>
4.&#160;Induction of &#945;-synuclein aggregates in primary embryonic dopamine neurons by seeding with preformed fibrils
Following any work with &#945;-synuclein pre-formed fibrils (PFFs), clean the laminar hood or any equipment that might have contacted the PFFs with 1% Sodium dodecyl sulfate (SDS), then with 70% ethanol.
<ol>
	<li>Prior to the experiment, dilute the PFFs with 1x PBS to a final concentration of 100 &#956;g/mL. Sonicate the diluted PFFs in microcentrifuge tubes with a bath sonicator at high power with water bath cooling at 4 &#176;C for 10 cycles, 30 s ON/30 s OFF. 
	NOTE: It is critical that the fibrils be properly sonicated to generate fragments ~50 nm long. The size of sonicated PFFs can be measured directly from transmission electron microscope images of stained PFFs. Sonication can be achieved as described above in a high-power bath sonicator. Alternatively, a tip sonicator can be used. Sonicated PFFs can be stored at -80 &#176;C in small aliquots to avoid multiple freezing/thawing cycles.</li>
	<li>On DIV8, add 3.75 &#956;L of 100 &#956;g/mL of PFFs per well to the 150 &#956;L of medium in the well to a final concentration of 2.5 &#956;g/mL. Use the same amount of 1x PBS for the control group.</li>
	<li>Prepare 4% paraformaldehyde (PFA) in 1x PBS and store the aliquots at -20 &#176;C. To do so, follow the steps below. 
	&#8203;NOTE:&#160;PFA is toxic; wear a mask and gloves during preparation, work always under a laminar hood, and dispose of all solid and liquid PFA waste according to the institution&#39;s directions.
	<ol>
		<li>Warm 500 mL of 1x PBS in a 1 L vessel. Put a stir bar in the vessel and put the vessel on a magnetic stirrer with a heating function. Adjust the temperature between 40&#8211;60 &#176;C to prevent boiling while keeping the solution warm.</li>
		<li>Measure 20 g of PFA powder under the hood in a disposable plastic measuring container. Carefully add the PFA powder into the vessel filled with 1x PBS. Start stirring the solution.</li>
		<li>Add 200 &#956;L of 5 M sodium hydroxide into the solution and continue stirring for ~15 min until the PFA dissolves completely.</li>
		<li>After the solution appears homogenous, add 168 &#956;L of 5 M hydrogen chloride to balance the pH to ~7. Check the pH with disposable color-fixed pH indicator strips.</li>
		<li>Remove the vessel from the heater and allow it to cool down to RT. Filter the solution and aliquot for storage at -20 &#176;C. Thaw the aliquots at RT before the use, and do not refreeze afterwards.</li>
	</ol>
	</li>
	<li>On DIV15, remove all media from the wells by pipetting. Add 50 &#956;L of 4% PFA to each well to fix the cells and incubate for 20 min at RT. After incubation, remove the PFA from the wells and add 100 &#956;L of 1x PBS to each well to wash the cells. Remove 1x PBS and wash 2x more.</li>
	<li>Leave 100 &#956;L of 1x PBS in each well to avoid drying. Store the plate at 4 &#176;C until immunochemistry is performed.</li>
</ol>
5.&#160;Immunofluorescent staining and automated imaging of primary embryonic dopamine neurons in 96 well plates
<ol>
	<li>Remove 1x PBS and permeabilize the cells by adding 100 &#956;L of 0.2% Triton X-100 in PBS (PBST) per well and incubating at RT for 15 min.</li>
	<li>Remove PBST and add 50 &#956;L of 5% normal horse serum (NHS) per well to the PBST. To block the unspecific antigen activity, incubate at RT for 1 h.</li>
	<li>Dilute the primary antibodies against tyrosine hydroxylase (TH) and pS129-&#945;syn (1:2,000) in 5% NHS in PBST. Add 50 &#956;L of diluted antibodies to each well and incubate overnight at 4 &#176;C.</li>
	<li>Remove antibodies and add 100 &#956;L of 1x PBS to each well to wash the cells. Remove 1x PBS and repeat washing 2x.</li>
	<li>To prevent the bleaching of fluorescent molecules, start working under minimum light conditions. Dilute the secondary fluorescently labeled antibodies (1:400) in PBST. Add 50 &#956;L of diluted antibodies to each well and incubate at RT for 1 h.</li>
	<li>Remove the antibody solution and add 100 &#956;L of 1x PBS to each well to wash the cells. Remove 1x PBS and repeat washing 2x.</li>
	<li>Remove 1x PBS, add 50 &#956;L of 200 ng/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) per well to stain the nuclei of the cultured cells, and incubate at RT for 10 min.</li>
	<li>Wash cells 3x with 100 &#956;L of 1x PBS for 5 min each. Keep 100 &#956;L of 1x PBS in each well after the last wash. Cover the plate with aluminium foil and store it at 4 &#176;C until imaging.</li>
	<li>Image primary embryonic dopamine neurons in a 96 well view plate with a high-content plate scanner (see&#160;Table of Materials) fitted with a 10x objective.</li>
	<li>Adjust the settings based on the specifications of the 96 well plate, such as plate type, manufacturer, size, distance between wells, as well as type and amount of medium.</li>
	<li>Select the imaging area of the well to cover all the cells in a micro island. Pick an example well to adjust the autofocus. Base the initial focus on DAPI.</li>
	<li>Calibrate the acquisition time for each fluorescent channel based on the intensity of the staining in control wells. Adjust the parameters so that in PFF-treated control wells, one can clearly distinguish dopamine cells harboring pS129-&#945;syn aggregates in cell soma, allowing for unambiguous quantification of pS129-&#945;syn positive and pS129-&#945;syn negative cells. 
	NOTE: Wells that do not contain PFFs should not have any staining for pS129-&#945;syn; therefore, these wells can be used as negative control for adjusting pS129-&#945;syn intensity.</li>
	<li>Image all the selected wells with a 10x objective simultaneously for all channels with immunofluorescence staining with exactly the same parameters.</li>
	<li>Optionally, label &#945;-synuclein inclusions in a subset of the wells with antibodies specific for filamentous &#945;-synuclein to confirm that changes in the number of pS129-&#945;syn-positive inclusions reflect the reduction in protein accumulation rather than inhibition of phosphorylation or dephosphorylation of pS129-&#945;syn.</li>
	<li>Repeat step 5.3, substituting pS129-&#945;syn antibody with &#945;-synuclein filament antibody (1:2,000). Image the stained aggregated &#945;-synuclein as in step 5.13.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% Trypan Blue stain</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml CELLSTAR Polypropylene tube</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M HCl</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>5 M NaOH</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>96-well ViewPlate (Black)</td>
			<td>PerkinElmer, Waltham, Massachusetts, USA</td>
			<td>6005182</td>
			<td></td>
		</tr>
		<tr>
			<td>99.5% Ethanol (EtOH)</td>
			<td>Altia Oyj, Rajam&#228;ki, Finland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AquaSil Siliconizing Fluid</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>TS-42799</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved 1.5 ml microcentrifuge tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Bioruptor sonication device</td>
			<td>Diagenode, Liege, Belgium</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+, Mg2+ free Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>CellProfiler and CellAnalyst software packages</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>Centrifuge 5702</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5702000019</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>HeraCell, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>BioRad Inc., Hercules, California, USA</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Ultra High Resolution Microscope with air table and cabinet</td>
			<td>GE Healthcare Life Sciences, Boston, Massachusetts, USA</td>
			<td>29254706</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease-I (Dnase I)</td>
			<td>Roche, Basel, Switzerland</td>
			<td>22098700</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>21331&#8211;020</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-mouse AlexaFluor 488</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-rabbit AlexaFluor 647</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-sheep AlexaFluor 488</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>A11015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>10500056</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Plain Economy PTFE Stir Bar</td>
			<td>fisher scientific, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>11507582</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageXpress Nano Automated Imaging System</td>
			<td>Molecular Devices, San Jose, California, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>25030&#8211;032</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-tyrosine hydroxylase</td>
			<td>Millipore, Merck KGaA, Darmstadt, German</td>
			<td>MAB318</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>17502&#8211;048</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Horse Serum</td>
			<td>Vector Laboratories Inc., Burlingame, California, United States</td>
			<td>S-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde powder, 95%</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-Fix, color-fixed indicator strips</td>
			<td>Macherey-Nagel, D&#252;ren, Germany</td>
			<td>92110</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospohate Buffer Saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>poly-L-ornithine</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, San Diego, California, USA</td>
			<td>ant-pm-1, ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit monoclonal anti-alpha-synuclein filament</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab209538</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit monoclonal anti-phospha-serine129-alpha-synuclein</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>RCT Basic, IKA Magnetic Stirrer</td>
			<td>IKA&#174;-Werke GmbH, Staufen, Germany</td>
			<td>3810000</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human glia-derived neurotrophic factor (hGDNF)</td>
			<td>PeproTech, Rocky Hill, NJ, USA or Prospec, Ness-Ziona, Israel</td>
			<td>450-10 (PeproTech), CYT-305 (Prospec)</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant mouse alpha synuclein Pre-Formed Fibrils (PFFs)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gift from collaborator, Prof. Kelvin C Luk</td>
		</tr>
		<tr>
			<td>Research Stereomicroscope System</td>
			<td>Olympus Corporation, Tokyo, Japan</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>sheep polyclonal anti-tyrosine hydroxylase</td>
			<td>Millipore, Merck KGaA, Darmstadt, German</td>
			<td>ab1542</td>
			<td></td>
		</tr>
		<tr>
			<td>TC 20 Automated Cell Counter</td>
			<td>BioRad Inc., Hercules, California, USA</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>11332481001</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>MP Biomedicals, Valiant Co., Yantai, Shandong, China</td>
			<td>2199700</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Er, S. et al., <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

<img alt="Figure 1" src="/files/ftp_upload/23098/23098_Figure1.jpg" /> 
Figure 1: Dissection of midbrain floor from E13.5 mouse embryo.&#160;(A) Cutting locations at the hindquarter of the head is marked with black arrows and white dashed lines. (B) The piece was removed from the rest of the embryo. The removed piece is circled. (C) The piece was turned 90&#176; to face the posterior towards the observer. (D<...

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...

Take micro-island cultures of primary embryonic mouse dopamine neurons in a multi-well plate containing media.
These neurons are pre-treated with pre-formed fibrils (PFFs) &#8212; misfolded and aggregated forms of the alpha-synuclein protein.
PFFs act as seeds, triggering further misfolding and oligomerization of endogenous alpha-synuclein, forming phosphorylated alpha-synuclein fibrils. These fibrils eventually form Lewy bodies.
Replace the media with a fixative to preserve cellular morphology.
Wash the cells with buffer.
Introduce a detergent-containing buffer to permeabilize the cell membranes.
Add a blocking solution to prevent non-specific antibody binding.
Add primary antibodies targeting phosphorylated alpha-synuclein and a reference protein ubiquitously expressed in dopamine neurons.
Wash to remove unbound antibodies.
Introduce fluorophore-conjugated secondary antibodies targeting the respective primary antibodies.
Wash again to remove unbound antibodies.
Add a DNA-binding dye to stain the nuclei.
Using a fluorescence plate scanner, quantify the dopamine neurons containing cytoplasmic alpha-synuclein aggregates.

13 조회수. Primary Embryonic Mouse Dopamine Neurons에서 알파-시누클레인 축적 연구

비디오: Primary Embryonic Mouse Dopamine Neurons에서 알파-시누클레인 축적 연구 - 실험

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

인간 A53T를 과발현 형질 전환 마우스의 뇌에 강화 된 ELISA에 의한 질병 관련 α-synuclein의 검출은 α-synuclein의 돌연변이

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

연구

JoVE Journal

의학

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

알파 - 시누 클레인 브릴의 주변 접종 후 형질 전환 마우스의 신경 염증의 생물 발광 영상

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

신경과학

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

자유 부동 면역 형광 분석에 의하여 절단 말초 신경 섬유에 있는 알파 Synuclein 응집체를 표적으로 하는

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

내레이션 대본

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons