Advice on sample preparation to improve your flow cytometry analysis of intracellular targets, focusing on the fixation and permeabilization steps. Some flow cytometry users are experienced with extracellular labeling with live or fixed cells. However, in order to measure expression of cytoplasmic proteins or post-translational modification of proteins involved in signaling pathways, additional protocol steps are required in order for antibodies to have access to their intracellular targets. Fixation cross-links macro-molecules in the cells and locks the target proteins in place, and permeabilization allows antibodies to access the intracellular space. This video goes over some of the considerations to keep in mind when choosing or testing reagents for fixation and permeabilization for your flow cytometry protocol.