Fixing time for IF 293 T cells - (Dec/01/2005 )

Inspired by another thread, I have one question.

How long would you suggest that I fix 293 T cells with 4 % PFA when they are grown on glass slides.

And how long would you permeabilize them with 0.2 % Triton X in PBS???

And pleez could you explain the difference of formaldehyde and paraformaldehyde. I'm used to fixing organs with PFA and I'm afraid my new lab only has 37 % formaldehyde (in H2O). Do I use it with 4 % in PBS as well???

Any other tips and tricks for 293 T and Immunofluorescence are welcome too!!!

Fix in 2-4% PFA in PBS for 15 minutes. Wash with PBS several times afterwards. GENTLY!

Permeabilize for 5-10 minutes (may depend on antigen and cells - 293T come off slides pretty quickly and easily so you may need to play around with the times and conditions for this part - be gentle!).

3.7% formaldehyde is essentially the same thing as 4% PFA which is the same thing as 10% neutral buffered formalin. Paraformaldehyde is just polymerized formaldehyde and many people use it as it gives a lot of flexibility as to what buffers one can use for fixation. Essentially the difference is just buffers. I do recommend though that you invest in some ampoules of 16% PFA. They are easier to use and less toxic than going into that nasty 37% formaldehyde bottle every time and exposing yourself.

Thankx a lot!!!

293 T are my favorite pets, so being gentle is my standard repertoire!!!

You are welcome, have fun with those little pets