Immunofluorescence Analysis of Cellular Localization

Cells were fixed in 3.7% paraformaldehyde, permeabilized on ice using 0.2% Triton X-100, and blocked using 3% BSA in phosphate-buffered saline (PBS). Samples were incubated in the indicated primary antibody for 1 hour at room temperature or 4°C overnight and another hour at room temperature with secondary antibodies (Alexa Fluor 594 α-rabbit, Alexa Fluor 488 α-mouse) (Life Technologies). Samples were mounted with Prolong Gold reagent containing DAPI (Life Technologies). Confocal images were acquired using a Fluoview 1000 laser scanning confocal microscope (Olympus Corp). Non-confocal images were acquired using an Eclipse Ti-U inverted laser microscope (Nikon). Quantification of total cellular signal and nuclear signal was conducted using ImageJ. Cells were normalized for both cytoplasmic and nuclear size and %nuclear signal was determined as previously described [57 (link)].