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