Feb 28, 2024
alpha-Synuclein pre-formed fibrils preparation and stereotactic injection into Mus Musculus brain
- 1University of Ottawa
Protocol Citation: Jean-Louis Parmasad 2024. alpha-Synuclein pre-formed fibrils preparation and stereotactic injection into Mus Musculus brain. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3pdjvzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
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Created: February 15, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95261
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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.
Storage
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)
PFF preparation (to be done on the same day as experiment)
On the day of usage, briefly thaw fibrils on ice.
2m
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.
2m
Program Covaris S220 water bath sonicator to:
18 W peak incident power;
20% duty factor;
50 cycles per burst;
150 s.
2m
Mouse preparation and administration
Mouse preparation and administration
Anesthetize mouse with isoflurane in an enclosed chamber.
2m
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.
2m
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
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.