This article details a protocol for immunofluorescence staining of skin biopsy sections from Parkinson's disease patients. The method focuses on detecting alpha-synuclein aggregates in dermal neurons, which are indicative of neurodegenerative conditions.
Take the cryosection of a skin biopsy from a Parkinson's disease patient encased in an embedding medium.
The tissue comprises an outermost epidermis, an underlying dermis, and subcutaneous fat innervated by nerves.
Wash the section to remove the embedding medium. Transfer it into a permeabilization-blocking solution, permeabilizing the cells and blocking non-specific binding sites.
Immerse the section in a primary antibody cocktail to achieve uniform antibody labeling, termed free-floating staining.
The antibodies enter the neurons and bind to alpha-synuclein aggregates distributed along the axons — an aggregated form characteristic of neurodegenerative diseases — and carboxyl-terminal hydrolases.
Introduce fluorophore-conjugated secondary antibodies that bind to the primary antibodies.
Treat with a fluorescent DNA binding dye that stains the nucleus.
Place the tissue on a slide, apply a mounting medium, and seal with a coverslip.
Under a confocal microscope, fluorescence from both antibodies reveals the presence of alpha-synuclein aggregates along the axons of dermal neurons.
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 a 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 4 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 anti-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 5 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 coverslip. Let the slides dry overnight. The next day, store the slides in an appropriate box at 4 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.
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