Co-localization of RASSF1A with microtubules. (A) SW-13/A (a, b &c ) or SW-13/AM (d, e &f) cells were grown on glass cover slips in medium containing 400 ug/ml G418 and after 24 hours, cells were fixed in cold Acetone-Methanol (1:1) for 10 minutes followed by immunofluorescence detection of cytoskeleton (using Rhodamine-Phalloidin; a &d) or RASSF1A (using anti-RASSF1A antibody and FITC-conjugated secondary antibody; b &e) or both (c &f). Cell nuclei fluoresces blue with DAPI. Arrows indicate areas of co-localization of RASSF1A and cytoskeleton (c &f). (B) RASSF1A-microtubule co-localization points in comparable number of photomicrographs of SW-13/A and SW-13/AM cells representing multiple views from duplicate experiments were manually counted, tabulated and presented as a graph. (C) Representative photomicrographs showing indirect immunofluorescence detection of RASSF1A and microtubules in the normal adrenal cortex (a) and ACC (b) tissue specimens. Red fluorescence represents microtubules, green RASSF1A and blue DAPI-stained nuclei. Arrows indicate co-localization of RASSF1A and microtubules. (Total magnification: 1000X).