If a component of the cytoplasm or nucleus has to be labeled, the plasma membrane must be permeabilized. There are several ways to perfom this, and they depend on the fixation method chosen. Cells fixed with solvents do not require additional permeabilizing steps - as the solvent has already extracted enough material out of the membrane. For this reason solvent fixation is doubly efficient. Cells fixed with cross-linking aldehydes need to have the plasma membrane integrity breached by the use of chemical agents. Commonly used reagents include DMSO and detergents like Triton X-100, saponin or deoxycholate.

Permeabilization is, like fixation, another area where fine tuning is necessary. Different detergents solubilize different molecules within the membrane, so it is necessary to know that the molecules you are interested in have not been washed away in the permeabilization step. The concentration of detergent is another detail that should be adjusted carefully. The idea is to selectively remove plasma membrane constituents to allow access to the cytoplasm without altering either the antigenicity or morphology of the sample.