I have a suspension cell, which cannot adhere to the surface. I had to use air-drying methods to perform my immunocytochemistry, with average result. I still need some more optimization. I'm also interested to combine the immunofluorescent with FISH to detect and localize my protein.
So, is there any negative effect of air-drying on the cells when I perform ICC together with FISH?
you can still prepare cytospins from your non-adherent cell suspension?
air drying probably won't interfere with FISH, while for immunocytochemistry the effect will be different regarding the antigen to be stained. we fix cytospins in cold methanol with good immuno results for different membrane, cytoplasmic and nuclear antigens as well as for FISH, I guess it will work also for the combination. regards
Irena
Hi,
IMO, air-drying is not good for cells prior to their fix (in any procedure): you can kindly pellet your cells, discard the surnatant, and then two or three passages with fixative (with the same procedure), leaving your cells in suspension. But, I've never done ICC with contemporary FISH. Have a good work
Simona
I think it is not a good idea to air-dry cells before fixation: your protein of interest may have degradation during drying. Once you cells, and wash it form fixer, you may air dry it. In this case you do not need premeabilization. Also you can easy make immunolocalization after fixation even in the eppendorff tubes, without any drying. For the combination with FISH, you have to use special pre-treatments. Good luck!
Hi Everyone, Thank you very much. Air drying is one of the method I tested. I also tested ICC without fixing and permeabilizing the cells with air drying and suspension method. Both give almost similar result. I will take note to fix them first and air dry after that to see the differences.For cytospins, still searching a lab that have the equipment. I'm 'isolated' in Chemical engineering department.
One advantage of air dry method is that you can work with very small number of cell and treat them after that. With suspension method, quite often, the pellet resuspend during supernatant removal and I have to spin them again...but usually it just lost forever.
I'm trying to isolate specific cells using cell sorter and the number of interested cell is around 1% of total population (still optimizing this one). One of the gated cell is around 80K cells of the tissue sample which is hardly seen after spinning. and still need to culture them.
Anyway, thank you very much for the tips and advice. I will try to come back if something interesting comes up.
Thanks again,
Fareed
You can dry cells on coated slide after fixation and washing in the tubes. You do not need to remove all supernatant, 10% of the liquid can remain and you wash 3-4 times with big volume of the buffer. You should use only bucket rotor for cell pelleting. Good luck!
Thanks again Taras Pasternak, I tried the 10% leftover and now I know why it did not work. I used fixed angle rotor. So, have to optimize other step which does not involved me asking a new rotor :)
I'm very interested with air-dry after fix/permeabilize method and will try it in near future.
Thanks again.
Yes, if cell completely air-dry on the slides, you do not need to permeabilized it. Just rehydrated in wet chamber for 20-25 minutes and add antibody thereafter.
Ooo...Now I understand what you try to tell me earlier. Correct me If I'm wrong. What you suggest is to let the rehydration of the cells (taking up water along with the antibody/RNA probes) to do the work without any additional permeabilization of the cells membrane. This may work with my current cells as the protein I'm interested with, localize under the cytoplasmic membrane with cytoplasmic bars a.k.a pores cells. But, If I want to target the internal protein, the cell still able to filter the antibody from coming in due to the ab size itself. That is why permeabilization of the membrane is needed. I may be wrong there.
Thank you.
Not exactly! When cell were completely drying, the part of the cell membrane should crack, with creating a big pores in membrane. Theoretically, you do not need to make additional permeabilization.
Hi guys, thanks for all the tips. After all the discussion and testing, I end up with another different method. I managed to perform ICC and FISH together and have them viewed under confoccal by immobilizing the cells in temperature sensitive hydrogel. Thanks again for all the tips.
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