An Immunofluorescence Assay to Detect Alpha-Synuclein Aggregates in a Skin Biopsy Section

Abstract

Source: Vacchi, E., et al. Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay. J. Vis. Exp. (2019).

This video demonstrates an immunofluorescence assay to detect neurodegenerative disease-specific alpha-synuclein aggregates on skin biopsy sections. Free-floating immunostaining is employed to fluorescently label the carboxyl-terminal hydrolase and the alpha-synuclein aggregates in the dermal neurons. Under a confocal microscope, cells with fluorescence from both antibodies indicate the presence of alpha-synuclein aggregates in the dermal neurons.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines,

1. Skin Biopsy Collection

1. Let a qualified physician perform the skin biopsy in an appropriate clinical setting.
2. Choose the area to perform the skin biopsy and clean it with an alcohol swab.
3. Prepare the anesthetic solution with 1 cc of lidocaine 2%.
4. With the needle parallel to the area, inject local anesthesia subcutaneously.
5. After checking the effect of anesthesia, take the 3 mm disposable punch and apply rotational and delicate downward pressure to rotate it d....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5G4 (anti human αSyneclein 5G4)	Analytik Jena Roboscreen	847-0102004001	Mouse monoclonal
AlexaFluor 488 Goat anti Rabbit IgG	Invitrogen	1971418	2mg/ml
AlexaFluor 594 Goat anti Mouse IgG	Invitrogen	1922849	2mg/ml
Disodium hydrogen phosphate solution	Merk Millipore	106586
Ethylene Glycol	Sigma-Aldrich	324558
Glycerol	Sigma-Aldrich	G7757
L-Lysine monohydrochloride	Sigma-Aldrich	L5626
Paraformaldehyde	Aldrich Chemistry	441244
PGP9.5	Abcam	ab15503	Rabbit polyclonal
Sodium Chloride	Sigma	S3014
Sodium Dihydrogen Phosphate Monohydrate	Merck Millipore	106346
Sodium (meta)periodate	Sigma-Aldrich	S1878
Trizma Base	Sigma	T1503
Tryton X-100	Sigma-Aldrich	X100
Vectashield	Vector Laboratories	H-1000	Mounting medium