Immunocytochemical localization of PI3K to cytoplasmic lipid bodies in U937 cells.

Cytoplasmic lipid bodies in U937 cells were stained with oil red O (A) or labeled with the fluorescent fatty acid, 1-pyrenedodecanoic acid (B). PI3K localization within cells was detected immunocytochemically with an mAb specific for the p85 isoforms of PI3K and avidin:biotinylated enzyme complex glucose oxidase immunocytochemistry, which yields colored reaction product at sites of p85 PI3K localization, including distinct punctate intracellular structures (C). In contrast, comparable immunocytochemistry with a control nonimmune mouse IgG yielded no staining (D). For dual labeling, U937 cells were labeled by incorporation of fluorescent fatty acid, 1-pyrenedodecanoic acid (E, G), and by immunocytochemistry with p85 mAb (F) or nonimmune mouse IgG (H). The punctate immunolocalization PI3K in (E) matched perfectly with fluorescent fatty acid–labeled lipid bodies (F). In contrast, although there were punctate fluorescent lipid bodies in control cells (G), no immunostaining was seen with nonimmune mouse IgG (H). It should be noted that fluorescent lipid body labelings in cells stained with PI3K (E) were weaker than in controls (G). This was likely due to quenching of fluorescence by the glucose oxidase product formed in the immunostaining (F) and the fact that larger lipid bodies in (H) were visualized as refractile, darker structures that lacked any specific glucose oxidase immunostaining. Objective magnification ×100 for (A) and (B) and × 63  for (C)-(H).