Technically, the normal intracellular staining protocols should work... Once the cells are porous, I won't think that there would be too much difference in getting the polyclonal in versus the secondary. We'll need more information as to what part are you trying to troubleshoot. Is the intracellular protein membrane bound, and if it is, to which organelle.

Hi Shen-An Hwang ! thanks for the answer, the protein im looking for is cortistatin, a emerging neuropeptide, because of that, the inmunedetection is horrible. Abs anti-cortistatin are not good.

You would do surface staining first followed by fixation and permeabilization then with you cortistatin.

If gene expression or protein detection are your end points you could always sort live CD4 and CD8 T cells then follow with qRTPCR or Western blot

http://www.proteinsimple.com/milo-flow-cytometry

Search the Protein Simple page for:

• Going with The Flow: Using Milo to Streamline Your Flow Cytometry Experiments. This is a technical note about How Milo can help you with flow targets. Very helpful.