An Immunofluorescence Assay to Detect Alpha-Synuclein Aggregates in a Skin Biopsy Section
Published: April 30, 2024
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 Let a qualified physician perform the skin biopsy in an appropriate clinical setting. Choose the area to perform the skin biopsy and clean it with an alcohol swab. Prepare the anesthetic solution with 1 cc of lidocaine 2%. With the needle parallel to the area, inject local anesthesia subcutaneo…
開示
The authors have nothing to disclose.
Materials
| 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 |
