Indirect immunofluorescence is a sensitive method to monitor the distribution of proteins. However, frequently a protein appears to be in distinct locations, even when analyzed in the same cell type under identical conditions. One possible cause that contributes to these discrepancies is the protocol used for immunodetection (1-3). Despite these complications, differences in staining can be useful to gain insight into protein functions associated with a specific subcellular location. The nucleoporin Nup98 is a compelling example for which the methodical comparison of staining protocols provides the basis to monitor its interaction with distinct compartments. We have chosen Nup98 as it is a mobile protein which resides in the cytoplasm, the nuclear interior, as well as the nuclear and cytoplasmic sides of NPCs (4-6). Within the nucleus, Nup98 associates not only with GLFG bodies in the nucleoplasm but also with nucleoli. Nup98 is a key component in nuclear transport (7-10) and an important factor in cancer research (11). As mislocalization of Nup98 fusion proteins correlates frequently with human leukemia, reliable methods are required that detect Nup98 in different locations. The protocol described here can be used to evaluate systematically and optimize the detection of proteins that are present in multiple cellular compartments.