We thank Parkinson Schweiz and ABREOC (the Scientific Research Advisory Board of the Ente Ospedaliero Cantonale) for their financial support of this study.

The protocol has been approved by the Cantonal Ethics Committee and all enrolled subjects gave written informed consent to the study.
1. Skin Biopsy Collection
<ol>
	<li>Let a qualified physician perform the skin biopsy in an appropriate clinical setting.</li>
	<li>Choose the area to perform the skin biopsy and clean it with an alcohol swab.</li>
	<li>Prepare the anesthetic solution with 1 cc of lidocaine 2%.</li>
	<li>With the needle parallel to the area, inject local anesthesia subcutaneou...

<ol>
	<li>McArthur, J. C., Griffin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+Tool+for+the+neurologist's+Tollbox.">Another Tool for the neurologist's Tollbox.</a> Annals of Neurology. 57, 163-167 (2005).</li><li>Wilkinson, P. F., Millington, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Skin. , 49-50 (2009).</li><li>Weddell, G., 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=Nerve+endings+in+mammalian+skin.">Nerve endings in mammalian skin.</a> Biological reviews of the Cambridge Philosophical Society. 30, 159-195 (1954).</li><li>Wang, L., 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=Protein+gene+product+9.5-immunoreactve+nerve+fibers+and+cells+in+human+skin.">Protein gene product 9.5-immunoreactve nerve fibers and cells in human skin.</a> Cell and Tissue Research. 261 (1), 25-33 (1990).</li><li>Holland, N. R., 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=Intraepidermal+nerve+fiber+density+in+patients+with+painful+sensory+neuropathy.">Intraepidermal nerve fiber density in patients with painful sensory neuropathy.</a> Neurology. 48, 708-711 (1997).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Archives of Neurology. 55 (12), 1513-1520 (1998).</li><li>Lauria, G., 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=European+Federation+of+Neurological+Societies/Peripheral+Nerve+Society+Guideline+on+the+use+of+skin+biopsy+in+the+diagnosis+of+small+fiber+neuropathy.+Report+of+a+joint+task+force+of+the+European+Federation+of+Neurological+Societies+and+the+Peripheral+Nerve+Society.">European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society.</a> European Journal of Neurology. 17, 903-912 (2010).</li><li>Provitera, V., 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=A+multi-center,+multinational+age-+and+gender-adjusted+normative+dataset+for+immunofluorescent+intraepidermal+nerve+fiber+density+at+the+distal+leg.">A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg.</a> European Journal of Neurology. 23, 333-338 (2016).</li><li>Wakabayashi, K., 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=Involvement+of+the+peripheral+nervous+system+in+synucleinopathies,+tauopathies+and+other+neurodegenerative+proteinopathies+of+the+brain.">Involvement of the peripheral nervous system in synucleinopathies, tauopathies and other neurodegenerative proteinopathies of the brain.</a> Acta Neuropathology. 120, 1-12 (2010).</li><li>Ruffmann, C., 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=Detection+of+alpha-synuclein+conformational+variants+from+gastro-intestinal+biopsy+tissue+as+a+potential+biomarker+for+Parkinson's+disease.">Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's disease.</a> Neuropathology and Applied Neurobiology. 44 (7), 722-736 (2018).</li><li>Lee, J. M., 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=The+search+for+a+peripheral+biopsy+indicator+of+alpha-synuclein+pathology+for+Parkinson+Disease.">The search for a peripheral biopsy indicator of alpha-synuclein pathology for Parkinson Disease.</a> Journal of Neuropathology &amp; Experimental Neurology. 76, 2-15 (2017).</li><li>Donadio, V., 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=Skin+nerve+misfolded+alpha-synuclein+in+pure+autonomic+failure+and+Parkinson+disease.">Skin nerve misfolded alpha-synuclein in pure autonomic failure and Parkinson disease.</a> Annals of Neurology. 79, 306-316 (2016).</li><li>Doppler, K., 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=Cutaneous+neuropathy+in+Parkinson's+disease:+a+window+into+brain+pathology.">Cutaneous neuropathy in Parkinson's disease: a window into brain pathology.</a> Acta Neuropathologica. 128, 99-109 (2014).</li><li>Zange, L., Noack, C., Hahn, K., Stenzel, W., Lipp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+alpha-synuclein+in+skin+nerve+fibres+differentiates+Parkinson's+disease+from+multiple+system+atrophy.">Phosphorylated alpha-synuclein in skin nerve fibres differentiates Parkinson's disease from multiple system atrophy.</a> Brain. 138, 2310-2321 (2015).</li><li>Braak, H., 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=Staging+of+brain+pathology+related+to+sporadic+Parkinson's+disease.">Staging of brain pathology related to sporadic Parkinson's disease.</a> Neurobiology of Aging. 24, 197-211 (2003).</li><li>Doppler, K., 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=Dermal+phospho-alpha-synuclein+deposits+confirm+REM+sleep+behaviour+disorder+as+prodromal+Parkinson's+disease.">Dermal phospho-alpha-synuclein deposits confirm REM sleep behaviour disorder as prodromal Parkinson's disease.</a> Acta Neuropathologica. 133 (4), 535-545 (2017).</li><li>Antelmi, E., 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=Skin+nerve+phosphorylated+&#945;-synuclein+deposits+in+idiopathic+REM+sleep+behavior+disorder.">Skin nerve phosphorylated &#945;-synuclein deposits in idiopathic REM sleep behavior disorder.</a> Neurology. 88 (22), 2128-2131 (2017).</li><li>Nolano, M., 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=Small+fiber+pathology+parallels+disease+progression+in+Parkinson+disease:+a+longitudinal+study.">Small fiber pathology parallels disease progression in Parkinson disease: a longitudinal study.</a> Acta Neuropathologica. , (2018).</li><li>Melli, G., 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=Cervical+skin+denervation+associates+with+alpha-synuclein+aggregates+in+Parkinson+disease.">Cervical skin denervation associates with alpha-synuclein aggregates in Parkinson disease.</a> Annals of Clinical and Translational Neurology. 5, 1394-1407 (2018).</li><li>Kovacs, G. G., 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=Intracellular+processing+of+disease-associated+alpha-synuclein+in+the+human+brain+suggests+prion-like+cell-to-cell+spread.">Intracellular processing of disease-associated alpha-synuclein in the human brain suggests prion-like cell-to-cell spread.</a> Neurobiology Disease. 69, 76-92 (2014).</li><li>Kovacs, G. G., 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=An+antibody+with+high+reactivity+for+disease-associated+alpha-synuclein+reveals+extensive+brain+pathology.">An antibody with high reactivity for disease-associated alpha-synuclein reveals extensive brain pathology.</a> Acta Neuropathologica. 124, 37-50 (2012).</li></ol>

The authors have nothing to disclose.

We describe a free-floating immunofluorescence assay for skin biopsies for the diagnosis of PD: it exploits double immunostaining with anti-PGP9.5 antibody, a panaxonal marker, and anti-5G4, a conformation specific antibody that recognizes the aggregated form of &#945;Syn.
The great advantages of skin biopsy for diagnostic purpose in PD and possibly in other protein conformational disorders are: 1) the direct access to nervous tissue prone to disease by a mildly invasive technique and thus wit...

Skin biopsy has acquired a great importance as the diagnostic and research tool in the field of neurological disorders1. Indeed, epidermis and dermis contain abundant somatic sensory nerve fibers (myelinated and unmyelinated), nociceptive free nerve endings, sensory receptors and autonomic innervation of sweat glands, vessels, sebaceous glands and muscle arrector pilorum2.
In the mid-20th century, the setup for immunohistochemistry of PGP9.5 antibody allowed the evidence of an extensive innervation of human epidermis mammalian skin3. PGP9.5 is a carboxyl-terminal hydrolase equally distributed along axons of both the central and peripheral nervous system (PNS). The availability of this antibody allowed not only to clarify the morphology and anatomy of PNS in the skin but also implemented the study of diseases associated with it3,4. Skin biopsy contributed to defining a new clinical entity: the small fiber neuropathy. Several international groups demonstrated the association between the loss of intraepidermal nerve fibers and symptoms/signs of small fiber neuropathy5 by skin biopsy analysis and provided standardized protocols for nerve morphometry as well as normative reference values to be used in the clinical practice6,7,8.
Recently a large amount of evidence has shown that neurodegenerative diseases, characterized by misfolded protein accumulations in the central nervous system, are multi-system pathologies9. Indeed, PD is characterized by &#945;Syn accumulation in the dopaminergic neuron of substantia nigra, but it has been demonstrated that &#945;Syn and its pathological form, phosphorylated &#945;Syn (P-&#945;Syn), could be detected also in the peripheral tissues. Gastro-intestinal mucosa10, salivary glands11, skin autonomic fibers surrounding sweat glands and pilomotor muscles12,13,14, show immunoreactivity to pathogenic forms of &#945;Syn, in accordance with Braak hypothesis that intriguingly postulate that &#945;Syn pathology may begin in PNS well in advance, before its accumulation in the brain15. Further, the presence of p-&#945;Syn has been demonstrated in skin nerves of patients with REM Behavior Disorders that are considered prodromal PD16,17 thus skin pathologic &#945;Syn can be considered a promising early peripheral histopathological marker of synucleinopathy.
The association of small fiber neuropathy in PD has been demonstrated previously and it has been found that intraepidermal nerve fibers density reflects disease progression18,19. Hence, the skin biopsy is a useful tool for studying neurodegeneration in PD and for establishing a pre-mortem histopathological diagnosis of the disease. Indeed, skin biopsy has a great advantage of being an easily accessible and minimally invasive procedure, allowing the analysis of nervous tissue prone to the pathology. Finally, the possibility of repeating the skin biopsy in the course of follow-up of the same patients allows studying the longitudinal correlation with disease progression.
In our laboratory, exploiting a double immuno-staining with PGP9.5 and the conformation-specific monoclonal 5G4 antibody, that recognizes disease specific forms of &#945;Syn including small aggregates20,21, we were able to show the presence of &#945;Syn aggregates in skin nerves with a promising high diagnostic efficiency19. Immunofluorescence analysis of the skin biopsy in conformational diseases stands out as a promising source of biomarkers by combining both the detection of protein aggregates and the measure of the neurodegeneration in vivo. Hereafter, we illustrate an easy and versatile protocol on handling the skin biopsy and performing the free-floating immunofluorescence staining for detecting &#945;Syn aggregates. Moreover, this protocol can be adapted for targeting any other protein of interest expressed in skin PNS.
The following study protocol has been used to evaluate the diagnostic utility of aggregated &#945;Syn analysis in the PNS of PD by skin biopsy19. Inclusion criteria for PD were: a definite clinical diagnosis according to the UK Brain Bank diagnostic criteria, disease duration at least 3 years, no family history, and no major cognitive impairment or major dysautonomic symptoms in the history. Exclusion criteria were known causes of neuropathy (glycated hemoglobin, creatinine, vitamin B12, TSH, serum immunofixation, HIV, HCV, syphilis, and borreliosis). Each subject underwent to 3 mm-diameter skin biopsies at three anatomical sites (neck at C8 dermatomal level, thigh 10 cm above the knee, leg 10 cm above lateral malleolus) on the side, which was clinically more affected. In general, the following protocol is about handling the skin biopsy and performing the free-floating immunofluorescence staining and analysis. Hence it can be adapted and used for the detection of other proteins of interest in skin tissue.

<table border="0" cellpadding="0" cellspacing="0" style="width:964px;" width="964">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">5G4 (anti human &#945;Syneclein 5G4)</td>
			<td style="width:161px;">Analytik Jena Roboscreen</td>
			<td style="width:344px;">847-0102004001</td>
			<td style="width:195px;">Mouse monoclonal&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 488 Goat anti Rabbit IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1971418</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 594 Goat anti Mouse IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1922849</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disodium hydrogen phosphate solution</td>
			<td>Merk Millipore</td>
			<td>106586</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Lysine monohydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>L5626</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>Aldrich Chemistry</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">PGP9.5</td>
			<td>Abcam</td>
			<td>ab15503</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride</td>
			<td>Sigma&#160;</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Dihydrogen Phosphate Monohydrate</td>
			<td>Merck Millipore</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium (meta)periodate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1878</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Base</td>
			<td>Sigma&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tryton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vectashield&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

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.

Following the described procedure (Figure 1), we detected &#945;Syn aggregates, labeled with 5G4 antibody, in dermal nerve fascicles innervating autonomic structures of PD patients. Morphology of alpha-synuclein deposits appears as a dotted signal along the axons of dermal nerves (Figure 2). Indeed, exploiting this protocol in 19 PD patients and 17 controls in skin biopsies at three anatomical site (cervical, thigh and distal leg) we found that 5G4 had 81% of se...

Watch this Scientific Journal Video about Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay at JoVE.com

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

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.

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

The diagnosis of Parkinson's disease still relies on clinical signs of motor involvement that appear later on in the course of the disease, when most of the neurons of the substantia nigra are already lost. This protocol allows the dermal nerves analysis by skin biopsy, that represents a potential new biomarker of the disease, to be used in clinical trials. Skin biopsy is an easily assessable, not-invasive procedure that allows the analysis of peripheral nervous tissue that is prone to the pathology.Alpha synuclein aggregates detection by skin biopsy can be used for diagnostic purposes, possibly at early phases, when potential cure is most effective. Follow-up skin biopsies in the same patient allow a time and special course analysis of alpha synuclein aggregate spreading in human cutaneous nervous system. These can shed light on the pathogenesis of disease.To begin this procedure, prepare a fresh PLP fixative solution as outlined in Table One of the text protocol. Immediately after collecting skin biopsy, submerge it in a tube containing 10 milliliters of the fixative solution. Incubate overnight at four degrees Celsius.The next day, in a fume hood, remove the PLP fixative gently. In the same tube, wash the biopsy three times for five minutes each, using five milliliters of 0.1 molar Sorensen's solution for each wash. Discard the Sorensen's solution, and add five milliliters of cryoprotectant solution.Incubate overnight at four degrees Celsius. The next day, store the biopsy at four degrees Celsius if the cut with a cryotome is to be performed within one week. First, set the cryotome to minus 20 degrees Celsius.Take a cryomold and fill it up with cryo-embedding medium. Using tweezers, immerse the biopsy into the cryo-embedding medium, with the longitudinal axis parallel to the bottom of the cryomold. Snap freeze the sample with liquid nitrogen, to obtain a solid cube of cryo-embedding medium containing the biopsy in the right orientation.Put the sample in the cryostat, and wait for 30 minutes to allow the biopsy to acclimatize. Then, fix the sample on the cryostat and cut into 50-micrometer sections. Using a small brush, transfer the cryo-sections to a 96-well plate containing 200 microliters of antifreeze solution in each well.After this, store the plate at minus 20 degrees Celsius. To begin, fill some of the wells of a fresh 96-well plate with 100 microliters of washing solution. Transfer the sections to be analyzed from the storage plate to the one containing the washing solution.Leave the sections in the washing solution for 10 minutes at room temperature. Then, transfer each section into an empty well containing the same washing solution, and repeat the wash. Next, add washing solution to the sections, and transfer them into new wells containing 100 microliters of blocking solution.Incubate at room temperature for at least 90 minutes, and a maximum of four hours. Dilute the primary antibodies, anti-PGP9.5 and anti-5G4, in the working solution. Transfer the sections into new wells containing 100 microliters of the working solution of the primary antibodies, and incubate overnight at room temperature.The next day, wash the sections in wells containing washing solution as previously described. Dilute the secondary antibodies, Goat anti-Rabbit to detect PGP9.5, and Goat anti-Mouse to detect 5G4, in the working solution. Transfer the washed sections into new wells containing 100 microliters of the working solution of the secondary antibodies.Cover the plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibodies, and incubate at room temperature for 90 minutes. After this, wash the sections two times in washing solution as previously described. Transfer the washed sections into new wells containing 100 microliters of DAPI for five minutes at room temperature.Wash the sections two times in washing solution as previously described. Then, mount the sections on a slide in the correct position, avoiding misfolding. Add a few drops of mounting medium to the slide and cover the section with a cover slip.Let the slides dry overnight. The next day, store the slides in an appropriate box at four degrees Celsius, making sure to avoid light exposure. Using an inverted fluorescence microscope or a confocal microscope, view the sections.Acquire the images by a camera connected to the microscope. Then, use a fluorescence microscope or a confocal microscope to analyze positive signals in sections in terms of spatial distribution and intensity of the signal as outlined in the text protocol. Following the described method, alpha synuclein aggregates labeled with 5G4 antibody are detected in dermal nerve fascicles innervating autonomic structures of PD patients.The morphology of alpha synuclein deposits appears as a dotted signal along the axons of dermal nerves. Representative use of this protocol in 19 PD patients and 17 controls in skin biopsies at three anatomical sites shows that 5G4 has a sensitivity of 81%and a specificity of 86%when compared to healthy controls. Phosphorylated alpha synuclein has a sensitivity of 56%and a specificity of 100%5G4 and phosphorylated alpha synuclein-positive deposits are found mainly around sweat glands, but are also found in muscle arrector pili, small vessels, and subepidermal and dermal plexus, but never in intraepidermal nerve fibers.Generally, within the sweat glands lumen, it is possible to observe a nonspecific signal that could be misinterpreted as 5G4 or phosphorylated alpha synuclein positivity. This type of signal is dotted, spherical, and it is due most probably to intraluminal autofluorescent material as demonstrated in technical controls without primary and secondary antibodies. Colocalization with PGP9.5 that marks the nerve fibers, which morphologically are filamentous and elongated, helps to identify the correct signal.Therefore, the specificity of 5G4 signal is highly increased by a double immunostaining with an axonal marker. An accurate fixation of the biopsy is necessary for producing a good-quality immunofluorescent staining and for the reliable interpretation of the fluorescent signals. If the fixative is not correctly prepared, or if an over-fixation has occurred, the result will be a high autofluorescence that will mask the signal of nerve fibers crossing the dermal-epidermal junction, or innervate the main dermal structures.The most critical step of this procedure is tissue fixation. Pay attention to the pH of PLP fixative solution, and timing of incubation, to avoid autofluorescence and a specific signal.

targeting-alpha-synuclein-aggregates-cutaneous-peripheral-nerve

Research

JoVE Journal

Neuroscience

8.0K visualizações.  Neurocenter of Southern Switzerland. O diagnóstico da doença de Parkinson ainda se baseia em sinais clínicos de envolvimento motor que aparecem mais tarde no curso da doença, quando a maioria dos neurônios da substantia nigra já estão perdidos. Este protocolo permite que a análise dos nervos dérmicos por biópsia da pele, que representa um potencial novo biomarcador da doença, seja usada em ensaios clínicos. A biópsia da pele é um procedimento facilmente avaliável, não invasivo que permite a análise do tecido ner...