Hi everyone, I have a problem with the ICC protocol for caspase-3 to detect the level of apoptosis. I am working with keratinocyte cells. Here is my previous protocol:
- I fixed the cell with 4% PFA for 15 min at 4 degree
- Wash with three times 5 min each
- Permeabilize the cell using 0.1% Triton-X in PBS for 20 min RT
- Then continued with blocking using 3%BSA in PBS for 1 Hour RT
- Primary antibody caspase-3 (1:100 dilution with blocking buffer) for overnight at 4 degree
- Wash with three times 5 min each
-Secondary antibody anti-rabbit 488 (1:250 dilution with blocking buffer) and DAPI for 1 hour RT
- Wash with three times 5 min each
However, it is really hard to clearly differentiate between apoptotic and non-apoptotic cells because almost all cells express it.
Do anyone happen to know a better protocol?
Thank you.
Hi Antonius Christianto
The protocol seems fine. Attachment of the images could have helped better to understand the possible errors in the protocol.
* Optimisation of the antibody dilution could help in reducing the overstaining of Casp-3. I would consider diluting it more and washing in between every step with 0.1 % T-X100 in PBS.
* Is the secondary antibody Alexa Flour 488? If so, then the recommended dilution is 1:1000 and DAPI staining should not be done along with secondary antibody since it can interfere with the fluorophores.
Hope it helps,
Thanks,
Hi Samir Ranjan Panda,
I have included an example of the result below for you to look at. I made a mistake: I used red fluorescent as a secondary antibody. As you can see, all seem to have positive signals.
Thank you very much for your suggestion. I'll try to do what you suggest.
Hello Antonius Christianto
Yes, I think DAPI is clearly overstained because of 1hr incubation. Please stain DAPI separately for 15mins at the end of secondary antibody. I do not think red fluorescence is an issue but you can try next time with Alexa 488.
Thank you,
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