immunostaining of neurons treated with alpha-synuclein aggregates

Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

To perform immunofluorescence in a chemical fume hood fix the neurons with 2% paraformaldehyde in PBS and 5% sucrose solution for 15 minutes at room temperature without shaking. After 15 minutes, remove the fixing solution, and briefly wash the neurons with PBS three times.
Next, permeabilize the neurons with 0.3% of nonionic surfactant in PBS for 5 minutes at room temperature. Then, briefly wash the neurons with PBS three times. Incubate the neurons with 3% FBS in PBS for 30 minutes at room temperature on an orbital shaker to block unspecific binding sites. Afterward, incubate the neurons with appropriate primary antibody dissolved in 3% FBS in PBS overnight in the cold room or refrigerator overnight at 4 degrees Celsius on an orbital shaker.
The next day, remove the antibody solution and wash the cells briefly with PBS for three times. Then, incubate the neurons with the appropriate fluorescent secondary antibody dissolved in 3% FBS in PBS for 1 hour at room temperature in the dark on an orbital shaker.
Remove the antibody solution and wash briefly with PBS for three times. Next, stain the neurons with DAPI solution for 15 minutes at room temperature in the dark on an orbital shaker. After 15 minutes, remove the antibody solution and wash briefly with PBS for three times. Mount the coverslips on the slide using anti-fade mounting medium.

immunostaining-of-neurons-treated-with-alpha-synuclein-aggregates

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1. Neurons Treatment
Note: The treatment has been performed at Division (DIV) 7. All the steps are carried on under a cell culture hood in sterile conditions. An example of cortical neuronal culture density at DIV 7 is illustrated in Figure 1.
<ol>
	<li>Pool microsomes-associated &#945;S aggregates obtained from the spinal cord of three different diseased Transgenic mice to have a 1 &#181;g/&#181;L solution, using the original homogenization buffer [composed of 250 mM sucrose, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM KCl (Potassium Chloride), 1.5 mM MgCl2 ( (Magnesium Chloride), 2 mM EDTA (Ethylenediaminetetraacetic acid), and 1x phosphatase/protease-inhibitors] for the dilution.</li>
	<li>Take a neuron culture on poly-D-Lysine coverslips in 24 well plates containing medium (2% B27, 10x gentamicin, and 2 mM glutamine). Remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 1x gentamicin, and 2 mM glutamine.</li>
	<li>Add 1 &#181;g of pooled microsomes-associated alpha-synuclein (&#945;S) aggregates to the cell medium. Return neurons to the incubator at 37 &#176;C.</li>
	<li>Every 3 days for 1 week, add 1/3 of fresh medium. Do not replace the medium. Just add it.</li>
	<li>After 1 week of treatment, remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 10 &#181;g/mL gentamicin, and 2 mM glutamine. Repeat every 3 days.</li>
	<li>Fix neurons after 2 weeks of treatment (DIV 21).</li>
</ol>
2. Immunofluorescence
<ol>
	<li>Fix neurons with 2% paraformaldehyde in Phosphate-Buffered Saline (PBS) 1x and 5% sucrose solution for 15 min at room temperature (RT) without shaking under a chemical fume hood.</li>
	<li>Remove the fixing solution. Briefly wash with PBS 1x, 3 times. 
	NOTE: Perform all the washing steps gently because primary neurons do not stick firmly to poly-lysine coverslips.</li>
	<li>Permeabilize neurons with 0.3% of non-ionic surfactant in PBS 1x for 5 min at RT.</li>
	<li>Briefly wash with PBS 1x, 3 times.</li>
	<li>Incubate neurons with 3% Fetal Bovine Serum (FBS) in PBS 1x for 30 min at RT to block unspecific binding sites on an orbital shaker.</li>
	<li>Incubate neurons with appropriate primary antibody dissolved in 3% FBS in PBS 1x, O/N at 4 &#176;C on an orbital shaker. 
	NOTE: syn303 (1:1,000), mouse &#945;S (1:200), pser129-&#945;S (1:1,000), and Tau (1:10,000) antibodies were used.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Incubate neurons with appropriate fluorescent secondary antibody dissolved in 3% FBS in PBS for 1 h at RT in the dark on an orbital shaker.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Stain neurons with DAPI (4&#8242;,6-diamidino-2-phenylindole) solution (0.1 &#181;g/mL in PBS 1x) for 15 min at RT in the dark on an orbital shaker.</li>
	<li>Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.</li>
	<li>Mount coverslips on a slide using an antifade mounting medium.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H0887-100ML</td>
			<td>1M pH=7-7.6</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>0390-100ml</td>
			<td>pH=8 0.5M</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-6X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>PhosStop</td>
			<td>Roche</td>
			<td>4906837001</td>
			<td>phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Euroclone</td>
			<td>EMPO14500</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX 4-20% Stain Free, 18 wells</td>
			<td>Biorad</td>
			<td>5678094</td>
			<td></td>
		</tr>
		<tr>
			<td>Supported Nitrocellulose membrane</td>
			<td>Biorad</td>
			<td>1620097</td>
			<td>0.2 &#956;m</td>
		</tr>
		<tr>
			<td>Blotting-Grade Blocker</td>
			<td>Biorad</td>
			<td>1706404</td>
			<td>Non-fat dry milk</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Termo Fisher Scientific</td>
			<td>34077</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Sigma-Aldrich</td>
			<td>1004411000</td>
			<td>65%</td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Termo Fisher Scientific</td>
			<td>1014355118NR1</td>
			<td>18 mm x</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Termo Fisher Scientific</td>
			<td>14170-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Termo Fisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/mL, 100 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Termo Fisher Scientific</td>
			<td>D5796-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Termo Fisher Scientific</td>
			<td>15400054</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Termo Fisher Scientific</td>
			<td>17504044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Termo Fisher Scientific</td>
			<td>35050-038</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Euroclone</td>
			<td>EC50182L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>1446600-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Termo Fisher Scientific</td>
			<td>15710</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Termo Fisher Scientific</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine arabinoside (AraC)</td>
			<td>Sigma-Aldrich</td>
			<td>C3350000</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Termo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>90 Ti rotor</td>
			<td>Beckman</td>
			<td>N/A</td>
			<td>Ultracentrifuge rotor</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syn-1 antibody, clone 42</td>
			<td>BD Biosciences</td>
			<td>610786</td>
			<td>anti-mouse WB: 1:5000</td>
		</tr>
		<tr>
			<td>Syn303 antibody</td>
			<td>BioLegend</td>
			<td>824301</td>
			<td>anti-mouse IF: 1:1000</td>
		</tr>
		<tr>
			<td>Tau antibody</td>
			<td>Synaptic Systems</td>
			<td>314 002</td>
			<td>anti-rabbit IF: 1:10,000</td>
		</tr>
		<tr>
			<td>pser129-&#945;S antibody</td>
			<td>A gift from Fujiwara et al, reference 19</td>
			<td></td>
			<td>anti-rabbit WB: 1:5000</td>
		</tr>
		<tr>
			<td>pser129-&#945;S antibody</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td>anti-rabbit IF: 1:1000</td>
		</tr>
		<tr>
			<td>Mouse &#945;S (D37A6) XP</td>
			<td>Cell Signaling</td>
			<td>4179</td>
			<td>anti-rabbit IF 1:200</td>
		</tr>
		<tr>
			<td>Alexa fluor 555-conjugated anti-rabbit antibody</td>
			<td>Termo Fisher Scientific</td>
			<td>A27039</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488-conjugated anti-mouse antibody</td>
			<td>Termo Fisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Microson XL-2000</td>
			<td>Misonix</td>
			<td></td>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>Ultra Bottles (Oakridge Bottles), PCB, 16x76mm, Assembly, Noryl Cap, Beckman-type</td>
			<td>Science Service EU</td>
			<td>S4484</td>
			<td>Ultracentrifuge tubes</td>
		</tr>
		<tr>
			<td>AXIO Observer Inverted Light Microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP2 laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted epi-fluorescence microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
			<td>Nonionic surfactant</td>
		</tr>
	</tbody>
</table>

Source: Panattoni, G., et al. <a href="https://app.jove.com/v/57884">Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation.</a> J. Vis. Exp. (2018)
This video demonstrates the immunostaining of neurons treated with alpha-synuclein (&#945;S) aggregates. The neurons are first treated with &#945;S aggregates. They are then fixed, permeabilized, and treated with a blocking solution. Primary antibodies are added, followed by a fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted and observed under a microscope.

<img alt="Figure 1" src="/files/ftp_upload/23012/23012_Figure1.jpg" /> 
Figure 1. Cortical neuronal cultures. Representative image showing density at DIV 7 of cortical neuronal cultures. Images were taken with an inverted light microscope, 10X objective. Scale bar = 100 &#181;m.

1. Neurons Treatment
Note: The treatment has been performed at Division (DIV) 7. All the steps are carried on under a cell culture hood in sterile ...

Begin with an adherent neuron culture treated with alpha-synuclein or alpha-S aggregates, which are clumps of a misfolded protein.
These clumps aggregate to form intracellular inclusions.
Replace the media with a fixing solution to preserve cellular integrity.
Remove the fixing solution and wash with buffer.
Next, add a surfactant to permeabilize the cellular membrane.
Remove the surfactant and wash with buffer.
Introduce a blocking solution to prevent non-specific antibody binding.
Now, incubate with primary antibodies that bind specifically to alpha-S inclusions and structural proteins.
Wash with buffer to remove unbound antibodies.
Incubate with fluorescently labeled secondary antibodies.
Remove unbound antibodies.
Add a fluorescent stain to label the nucleus.
Remove the stain and wash with buffer.
Using mounting media, mount the coverslip with neurons on a slide.
Under a fluorescent microscope, observe immunolabeled alpha-S inclusions within the neuron.

44 visualizações. Imunomarcação de neurônios tratados com agregados de alfa-sinucleína

Vídeo: Imunomarcação de neurônios tratados com agregados de alfa-sinucleína - Experimento

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Extração seqüencial de solúveis e insolúveis alfa-sinucleína de Brains parkinsonianos

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

JoVE Journal

Neurociência

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.

Imagem de bioluminescência de Neuroinflammation em Ratinhos Transgénicos Após inoculação periférica de alfa-sinucleína fibrilas

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

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.

Segmentação de agregados Alpha synuclein em fibras nervosas periféricas cutâneas por ensaio de imunofluorescência livre-flutuante

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

Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

Transcrição

Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates