Dear All I am trying to detect kinetochore localization of MAD2 induced by a microtubule depolymerizing agent in SW480 human cancer cells. I have used 2 different protocols (-20 oC 100% methanol fixation for 20 minutes or 4% formaldehyde fixation for 20 minutes, both followed by permeabilization with either 0.3% triton/PBS or 100 micog/mL digitonin/PBS) but unfortunately I got negative results. I had success with microtubules immunocytochemical staining using methanol fixation method and I demonstrated the induction of prometaphase arrest by my treament, but it seems that MAD2 immunostaining is not as easy as microtubules. I would greatly appreciate if anyone has an experience in MAD2 immunocytochemical staining provides me with a help. thanks a lot Regards, Yobou I.G., M.Sc. PhD graduate student kyoto prefectural university of medicine Japan
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