Dear all,I am optimizing stainings of a soluble protein of 6-7 KDa molecular weight. I use 4% PFA for fixation and 0.3% Triton x100 for permeabilization. I am wondering if somehow my protein of interest comes out of the cells during permeabilization? Could anyone suggest a protocol to avoid the "probable leakage" of this soluble protein from cells during permeabilization and subsequent stages. Some people suggest saponin for permeabilization. Does any one has  suggestions of different fixation and permeabilization (saponin concentrations or others) for the  soluble proteins.

Many thanks,

Faisal