Apr 02, 2023

Public workspaceAlpha-synuclein immunochemistry on STC-1 cells using DAB

DOI
dx.doi.org/10.17504/protocols.io.kqdg39yjpg25/v1
  • 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland, USA.
Michael J Hurley
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DOI: dx.doi.org/10.17504/protocols.io.kqdg39yjpg25/v1
Protocol Citation: Michael J Hurley 2023. Alpha-synuclein immunochemistry on STC-1 cells using DAB. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39yjpg25/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: February 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 77692
Keywords: ASAPCRN, STC-1, immunohistochemistry, alpha-synuclein
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
This protocol describes how to visualise alpha-synuclein in STC-1 cells by DAB immunohistochemistry. It also works for other antibodies (e.g. 5-HT, CCK, GLP).
Materials
Rabbit anti-alpha-synuclein ab212184 Abcam

Rabbit anti-beta-actin ab241153 Abcam

Goat anti-rabbit IgG biotin 111-065-144-JIR Stratech

Streptavidin POD11089153001 Roche
Alpha-synuclein immunochemistry on STC-1 cells using DAB
Alpha-synuclein immunochemistry on STC-1 cells using DAB
1d 0h 58m
1d 0h 58m
Grow STC-1 cells on 12 mm glass coverslips treated with poly-D-lysine or Geltrex™.
Fix cells with 4 % paraformaldehyde Duration00:15:00

Safety information
Paraformaldehyde is toxic by inhalation
158127 (sigmaaldrich.com)

15m
Wash cells with PBS 2 x Duration00:05:00

5m
Aspirate and discard PBS and freeze cells < Temperature-20 °C

Thaw frozen 24-well plates containing fixed cells grown on glass coverslips
Wash cells with PBS Duration00:05:00

5m
Quench with 0.3% H2O2 Duration00:05:00

5m
Wash cells with PBS 2 x Duration00:05:00

5m
Block with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosalDuration01:00:00

1h
Incubate with rabbit anti-alpha-synuclein (1:500) (Abcam, ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (block buffer) Duration20:00:00

20h
Wash with PBS 4 x Duration00:05:00

5m
Incubate with biotinylated goat anti-rabbit IgG (1:500) (Stratech, 111-065-144-JIR) in 2 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) Duration02:00:00

2h
Wash with PBS 4 x Duration00:05:00

5m
Incubate with 1:250 streptavidin-HRP (Roche, 11089153001) for 1 hour in antibody bufferDuration01:00:00

1h
Wash with PBS 6 x Duration00:05:00

5m
Visualise staining by incubation with PBS containing 0.05 % (w/v) 3,3′-Diaminobenzidine/0.015 % (v/v) H2O2/0.05 % (w/v) nickel ammonium sulphate for ≤ Duration00:03:00

3m
Rinse for > Duration00:05:00 in running tap water

5m
Remove coverslips from wells and mount on microscope slides with Hydromount™ (National Diagnostics)