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I would like to request some help for ICC protocol for the Anti-Cre recombinase antibody (ab190177).
There is picture on the website which represent a ICC detection of the Cre protein.
Could you tell me what were the exact steps for the ICC protocol? For example: what fixating/blocking/permeabilizating agents were used and what were the parameters of the incubations?
Waiting for your reply!
Asked on Jul 30 2015
Thank you for your enquiry.
I contacted the originating laboratories who have provided the following ICC protocol.
-Fixate with PBS with 3% paraformaldehyde for 10 min @ RT
-Permeabilize cells for 2 min with cold methanol.
- Wash 3x with PBS.
- Block the cells in 2% BSA-PBS-0.2%Tween (BSA-PBS-T) 1h @ RT
- Incubate with anti-Cre (diluted 1:2000 up to 1:4000) in BSA-PBS-Tween overnight @ 4C
- Wash 3-4X with PBS-0.2% Tween
- Incubate with secondary in BSA-PBS-Tween 1 h @RT
- Wash 3-4X with PBS-T
- Wash 2X PBS only
- Rinse in water
I hope this will be helpful. Please note that this may require some individual optimization.
Sam WasherAbcam Scientific Support
Answered on Jul 30 2015