Aug 28, 2024
Fluorescent Immunolabelling for Alpha-Synuclein in neuronal primary culture (Testing PFF toxicity)
- Sabina Marciano1,2,
- Rong Chen3,2,
- Ted Dawson4,2,
- Roberta Marongiu1,2
- 1Department of Neurological Surgery, Weill Cornell Medical College, New York, NY 10065;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Johns Hopkins University;
- 4John Hopkins University School of Medicine
Protocol Citation: Sabina Marciano, Rong Chen, Ted Dawson, Roberta Marongiu 2024. Fluorescent Immunolabelling for Alpha-Synuclein in neuronal primary culture (Testing PFF toxicity). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzr6wv8j/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
We use this protocol and it's working
Created: August 23, 2022
Last Modified: September 23, 2024
Protocol Integer ID: 69056
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
This protocol is designed to perform fluorescent immunolabelling on neuronal primary culture after PFF incubation.
The labelling of phosphorylated Alpha-synuclein is considered as a marker of the PFF toxicity and should be performed to test the PFF before in vivo injection.
See protocol for PFF preparation
and protocol for neuronal primary culture
Fluorescent immunolabelling of phosphorylated Alpha Synuclein (green) in neuronal primary culture.
Image Attribution
image by Sabina Marciano
Materials
Reagents:
Purified anti-α-Synuclein Phospho (Ser129) AntibodyBioLegendCatalog #MMS-5091
Anti-MAP2 antibody (ab5392)AbcamCatalog #ab5392
Donkey Anti-Mouse IgG H&L (Alexa Fluor® 488) AbcamCatalog #ab150105
Donkey anti-Chicken IgY (H L) Highly Cross Adsorbed Secondary Antibody Alexa Fluor™ 594Invitrogen - Thermo FisherCatalog #A78951
Solutions:
4% (wt/vol) Paraformaldehyde/4% (wt/vol) sucrose/PBS
For 50 ml in PBS, pipette 10 ml of 20% paraformaldehyde into ~40 ml of PBS, add 2 g of sucrose and mix it until it dissolves. Bring the volume to 50 ml with PBS. This solution works best if it is used at room temperature, although others report optimal results when it is warmed to 37degrees. This solution can be stored at 4 degrees celsius, protected from light, for 1 week.
PBS/BSA0.005%/TWEEN0.05%
Dissolve 1mg BSA in 10mL PBS. Use 10uL of this solution for 20mL of PBS. Add 10uL Tween 20. Prepare this fresh on the day of use. All the washing steps are done for 10min.
Aspirate the medium from coverslips (aspirate from only a few coverslips at a time).
Add 4% (wt/vol) PFA /4% (wt/vol) sucrose (1 ml for a 12-well plate).
Incubate the plate at Room temperature for 00:15:00 .
15m
Wash 3 times 00:10:00 with PBS/BSA/TWEEN solution.
10m
Proceed or store in the fridge in PBS, protected from light, for up to 1 week.
Set up humidifier chambers.
Incubate with 0.5% Triton in PBS/BSA/TWEEN for 00:05:00 .
5m
Wash 3 times 00:10:00 with PBS/BSA/TWEEN.
10m
Block in Donkey Serum 1:20 in PBS for 00:30:00 .
30m
During the blocking, prepare the primary antibody solution.
In PBS/BSA/TWEEN, add Biolegend psyn (MMS-5091) Mouse (1:1000) and Abcam MAP2 (ab5392) chicken Millipore (1:2000).
Incubate with primary antibody for 01:00:00 at Room temperature .
1h
Wash 3 times 00:10:00 with PBS/BSA/TWEEN.
10m
During the washes, prepare the secondary antibody solution:
In PBS/BSA/TWEEN, add Alexa Fluor 488 donkey anti-mouse (1:1000) + Alexa Fluor 594 donkey anti-chicken (1:1000).
Incubate in secondary antibody solution for 01:00:00 at Room temperature in the DARK.
1h
Wash 3 times 00:10:00 with PBS/BSA/TWEEN.
10m
Incubate with DAPI 1:10,000 in PBS 00:10:00 at Room temperature .
10m
Wash 3 times 00:10:00 with PBS.
10m
Mount the coverslips onto glass slides with Prolong Gold mounting medium.