Jun 27, 2024
IMMUNOCYTOCHEMISTRY PROTOCOL
- Scott Vermilyea1
- 1University of Minnesota
Protocol Citation: Scott Vermilyea 2024. IMMUNOCYTOCHEMISTRY PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnorygk5/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: June 27, 2024
Last Modified: June 27, 2024
Protocol Integer ID: 102542
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details about the Immunocytochemistry.
Procedure
Procedure
1h 30m
Select coverslips previously fixed in 4% paraformaldehyde and subsequently washed and stored in PBS solution.
Permeabilize: 0.2% TX-100 in PBS for 00:15:00 at Room temperature .
15m
Wash 3 x 5 min in PBS.
Wash 00:05:00 in PBS. (1/3)
5m
Wash 00:05:00 in PBS. (2/3)
5m
Wash 00:05:00 in PBS. (3/3)
5m
Block: 50% Sniper in PBS for 00:15:00 at Room temperature .
15m
Wash 3 x 5 min in PBS.
Wash 00:05:00 in PBS. (1/3)
5m
Wash 00:05:00 in PBS. (2/3)
5m
Wash 00:05:00 in PBS. (3/3)
5m
Primary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100 overnight at 4 °C (gentle rocking).
Wash 3 x 5 min in PBS.
Wash 00:05:00 in PBS. (1/3)
5m
Wash 00:05:00 in PBS. (2/3)
5m
Wash 00:05:00 in PBS. (3/3)
5m
Secondary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100.
- Can add DAPI in secondary solution or as a separate step following washing of the secondary.
Wash 3 x 5 min in PBS.
Wash 00:05:00 in PBS. (1/3)
5m
Wash 00:05:00 in PBS. (2/3)
5m
Wash 00:05:00 in PBS. (3/3)
5m
Mount coverslips to slide with Vectashield antifade mounting medium.