I am doing a immunofluorescence imaging of my suspension cells (EBV lymphocytes) to look for RAD51. However for some reason I am not able to see any RAD51 in the cells. The DAPI stain works but not able to visualize the RAD51 signal. I used the same protocol for HEK293T cells and U2OS cells and it worked, but it isn't working with the suspension cells. My protocol is as follows:
1. Treat the cells with MMC.
2. Release and collect the cells.
3. Wash the cells with PBS - centrifugation.
4. Fix the cells in PFA - 10 min - RT
5. Wash the cells with PBS - centrifugation.
6. Permeabilize the cells in 0.1% TritonX in PBS - 15 min - RT.
7.Block in 5% BSA (in 0.1% Triton X in PBS) - 1 hour - RT.
8. Incubate in primary Ab - overnight at 4C.
9. Wash with 0.1% Triton X in PBS - 3 times.
10. Incubate in secondary Ab - 1 hour - RT.
11.Wash with 0.1% Triton X in PBS - 3 times.
12. Add DAPI+mountant and mount on slides.
Is there something that I change or avoid ?
Thank you
If you use the same protocol for the other two cell types and it works there you might not have a detectable amount of RAD51 in your lymphocytes. Did you check with a different method (e. g. westernblott) if and how much RAD 51 is there?
The only step I do differently is the fixation, where I incubate for at least 15 minutes in PFA. Then you can be sure that also soluble intracellular protein is fixed and does not get washed away in the following steps.
Good luck! Best regards
Tilo
I can not see anything wrong in your protocol, similar to mine, and the no signal staining is very common to me as well, the main problem is the Ab. Whatever the supplier boast the Ab can do on their website, it is very normal you can not repeat. To save your time and efforts, change a validated Ab will be a good choice. Good luck.
-Your protocol is very similar to mine except that i fix my cells in methanol at -20 for at least 10 minutes. Then i wash and i directly cytopsin cells on slides.
-if you were able to detect RAD51 in other cell lines i don't see a problem with the antibody.
- you should check by other techniques if your lymphocytes express RAD51.
Thanks all ! My answers for questions raised above:
I have used the same Ab for HEK293-T cells and it works. It is just not working with the lymphocytes.
I checked the literature and see that people show IF images of lymphocytes stained for RAD51.
I am trying 15 min fixing with PFA, lets see if this works.
I am also including a control with HEK-293T cells, will keep you informed.
I am trying with more controls this time, and hope it works. If it does not, then I am thinking to do a western blot to confirm the levels of RAD51.
Will keep this thread posted. Thank you again for the inputs.
Sounds good! But don't trust the literature too much, check if YOUR lymphocytes have RAD51 by an independent method.
And don't forget to upvote our answers if you found them useful :)
Good luck, I hope it works!
Hello All!
An update! I included HEK293 controls this time and also my WT lymphocyte control. Both of them seem to have foci (I say seem because apparently I did not wash the cells enough after Ab binding since I was having few cells to work with and did not want to lose them all, hence the Non treated controls also have some foci which I think is because of not adequate washing). My Mutant-Mutant and Mutant-wild type lymphocytes did not show any foci and my Wild type lympho and HEK293 showed foci. I am re-doing the experiment to get rid of the foci in the non treated by proper washing. These are the things I did differently this time:
1. Increased my fixing time to 15 minutes. (Previously 10 minutes)
2. Incubated my cells in primary Ab overnight at 4C with rotation. (previously without rotation).
Hope it works this time again and then I can be confident.
Thanks all !
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