PTEN is a well-known tumor suppressor with various functions that depend on its intracellular localization. Green fluorescent protein (GFP)-tagged live-cell images clarified the crucial amino acids needed to regulate the localization of PTEN in cells. However, it currently remains unknown whether GFP itself affects the intracellular localization of PTEN and its mutants, and the establishment of fixed-cell imaging is important for identifying the exact location of PTEN in cells. I herein investigated a number of immunofluorescence strategies for cell fixation, membrane permeabilization, and antigen retrieval. Permeabilization by detergents was necessary to observe nuclear and cytosolic PTEN in paraformaldehyde (PFA)-fixed cells; however, this permeabilization was not always valid. On the other hand, antigen retrieval by the pre-boiled EDTA treatment was useful for detecting plasma membranous PTEN in PFA-fixed cells in the same manner as in in vivo studies. Furthermore, methanol-fixed images of PTEN were consistent with GFP-tagged live-cell images. Two immunofluorescence methods (the PFA-fixed/pre-boiled EDTA treatment and methanol fixation) are applicable to investigations of the intracellular localization of PTEN without a GFP tag in cultured cells. In conclusion, live-cell imaging and appropriate immunofluorescence including a novel antigen retrieval treatment were both useful for detecting the cellular localization of PTEN, particularly at the plasma membrane.