you can store them a while in PBS at 4°C after fixation. But I would not let it longer than a week, at least for PFA fixation. For acetone, Methanol, use them directly, if possible. You store the fixation solution at -20°C, right. It will not freeze! You add it directly to your cells (washed before slightly with warm PBS to remove media stuff). I even put my cells for 5 min at -20°C for fixation. Good luck, Geraldine

I haven't done tight junction proteins before, but I seem to remember from friends doing it that PFA is better at preserving them then alcoholic fixatives. I'm sure someone will confirm or deny this. As far as Ac/MeOH goes, it's a great fixative for lots of things. No, it will not freeze, you use it straight from the freezer. I've always kept my cells at room temperature whilst fixing, but I know people get good results with fixing in the freezer as well. Be aware that the solution is pretty volatile, so add plenty of fixative to your wells and keep your lid on, especially if fixing at room temperature.

I would advise against storing the cells for any extended period of time before staining. If you do store them, yes PBS at 4°C is appropriate. I have been told in the past to add a bit of protein to the PBS (BSA or serum) to improve preservation, but don't know if this actually does anything.

Thank you very much Geraldine and Niels for your responses. I really appreciate them all.

I made a quick survey on journals studying tight junction proteins by immufluorescence and found out that both PFA or alcoholic fixatives produce good images. I chosed ice cold acetone:methanol (1:1) fixation method just to skip permeabilization step.

Are you planing to do FACS, this may be a good reason for keeping them in suspension. But if you are going to be looking thorough a microscope, one approach is to fix them on the slide. To do this, wash you cells in buffer. Then  you should put a drop of your cells on a "+" slides ( which have a coating which helps the cells stick). Give them time to stick but do not let them dry out. After about  2-10 minutes, rinse them in buffer and go directly into your fix.  You can chill it .(About 10-20 secs). Then let them dry out. These slides can then be stored at -20 or -70C.  Place them in an air tight slide box with dye rite. You will be able to store them for a long time.

There is also a modification of this fix. You can add 5 cc of 10% formalin (use the saturated formaldehyde solution to make a "10 %" solution.)  This fix  can be used in the same way. but after fixation, rinse in water and then let the slides dry. This may be a good compromise fix for what you are looking at, as you may not need to do any permeabilzation  but you have included some formalin. Good luck

I've used methanol-acetone fixation protocols for sperm cells, and they included treating the air-dried smear first with cold methanol and then with cold acetone. I don't know, however, why the sources recommended the two fixatives to be used sequentially; and anyway, your object is very different.