alpha-Synuclein pre-formed fibrils preparation and stereotactic injection into Mus Musculus brain
Jean-Louis Parmasad
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Abstract
Preparation of seed-competent alpha-Synuclein pre-formed fibrils and subsequent stereotactic injection into Mus Musculus brain as a model of synucleinopathy.
Steps
Storage
Store alpha-Synuclein fibril preparations at -80°C with appropriate biohazard precautions and labels until use.
PFF preparation (to be done on the same day as experiment)
On the day of usage, briefly thaw fibrils on ice.
Transfer required quantity of fibrils to the appropriate tube compatible with your sonicator. Dilute fibrils with normal saline (0.9% NaCl) if required. We used fibrils at a concentration of 5mg/mL.
Program Covaris S220 water bath sonicator to:
18 W peak incident power;
20% duty factor;
50 cycles per burst;
150 s.
Mouse preparation and administration
Anesthetize mouse with isoflurane in an enclosed chamber.
Once deeply anesthetized, transfer the mouse to the stereotaxic frame. Insert mouse nose into isoflurane mask.
Using a 2µl Hamilton Company syringe, take up 1µl of sonicated PFFs or normal saline (0.9% NaCl) using a micro-syringe computerized pump.
Program the micro-syringe computerized pump to deliver 1 μL mouse PFFs (5 mg/mL) or sterile saline (0.9% NaCl) at a rate of 0.1 μL/min (min).
Align needle to coordinates relative to bregma: −2 mm medial-lateral; +0.2mm antero-posterior.
Incise skin. Mark site on skull with a marking pen, and drill 1mm diameter burr hole with handheld drill.
Slowly position the needle to coordinates relative to bregma: −2 mm medial-lateral; +0.2mm antero-posterior and −2.6 mm dorso-ventral (right dorsal striatum).
Begin the administration sequence, following the micro-syringe computerized pump's instructions.
Leave the needle in place for at least 3 min before its slow withdrawal.
Close skin with topical skin adhesive.
Post-injection
Provide appropriate post-surgical care to the mice.
Wait 6 months for phenotype development, or however long your experiment requires.
Perform behavioural experiments then collect tissues for biochemical and histological analysis.
