Aggregation propensity of α-synuclein seeds in primary neurons
Arpine Sokratian
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Abstract
This protocol describes the details of the treatment of primary hippocampal culture with α-synuclein fibrils or monomer protein. It also includes details of the collection of cell lysates for immunoblotting or ELISA measurements. Another part of the protocol describes immunostaining analysis of treated neurons to measure the seeding propensity of α-synuclein fibrils visualized with phospho-S129 α-synuclein antibodies.
Steps
Prepare primary hippocampal neurons according to protocol
Preparation of primary hippocampal neurons
at DIV7 treat the culture with α-synuclein fibrils/seeds or monomeric protein at concentration 0.64 nM or 64 pM relative to estimated molecular weight measured as calculated size by DLS acquisitions and protein weight denatured in 6M GuHCL and calculated via Nanodrop.
Dynamic Light Scattering measurements
At DIV21 for 14 days of incubation or at DIV14 for 7 days of incubation culture media should be removed - aspirate the media
Here are options to use the prepared cultures for the western-blot analysis or ELISA (step-case) or for the immunofluorescence
Spin down a tube of normal donkey serum (20k xg for 10 min, 4C). Remove supernatant, do not use pellet.
Rinse with 3x with TBS. Use plastic bulb dropper and be careful. Prefer to remove w/ 200uL pipette, not vaccum. DO NOT let cells dry out, only do a row at a time.
Block/ Permeabilize with 300 uL 0.1% saponin, 3% Normal Donkey serum in TBS 30 min RT [KEEP COVERED]
During block, prepare primary antibodies:
Add primary antibody at desired concentration with a supplement of 0.02% saponin, 1% donkey serum, in TBS. Spin down the tube (20k xg for 10 min, 4C). Remove supernatant, do not use pellet
MJFR14 (EPY, 1:4,000), Tau5 (1:2,000), NeuN (1:2,000)
Remove block from wells.
Add primary antibody solutions to well. (300uL)
Overnight at 4C. Gentle rocking (no orbital shaker). Cover in Foil and make sure no loss of liquid.
Rinse with 1x with 300uL of TBS
Prepare secondary antibodies.
Use anti-rabbit AlexaFlour 647, anti-mouse AlexaFlour 488, anti-chicken AlexaFlour 555 at 1:1000 concentration, supplemented with 0.02% saponin, 0.1% normal goat serum in PBS. Spin down the tube (20k xg for 10 min, 4C). Remove supernatant, do not use pellet. Add Hoescht 1:10,000 to staining buffer
Rinse two times with TBS
Add TBS imaging buffer, take pics on Keyence.
Imaging: let the plate come up to RT, wipe bottom of plate with 70% ethanol before imaging
Take x25 20x pictures per well.
Images were obtained using Keyence BZ-X810 and Zeiss880 and coded for a blinded approach to analyze using CellProfiler and imageJ.
