Immunofluorescence - the role of buffers with and without Calcium and magnesium (Sep/18/2008 )

Hi,

I was wondering what kind of bufferes are the best to use for washing and preparing Ab dilutions for immunofluorescence.

I have both PBS with calcium and magnesium and also PBS without calcium and magnesium. I am trying to look for membrane proteins.

Thanks

Pooja

PBS with Ca and Mg is used to keep the cells attached to the coverslip. Some cells detach easily so PBS with CA and Mg is used.

If your cells donot detach easily, then use PBS without Ca and Mg. This should not affect the immunofluorescence for any particular protein.

Thanks for the information.

Also I had another question. The protocol that I am following includes the 4% formaldehyde fixation procedure.... but with 2% sucrose in it. what is the role of sucrose?

Pooja

depends on the protocol, but I don't use Sucrose, just add 4% paraformaldehyde and then permeablize with a Triton X100 solution.