Subcellular Localization of Ago2 Protein

TIVE cells were grown overnight on coverslips at a dilution of 1 x 10⁴ cells per well in a 6-well plate. Nuclei isolated from PEL cells were prepared as described [72 (link)], and fixed with a 1:1 ratio of methanol and acetone for 10 min in a humid chamber at 4 ˚C. The samples were blocked in PBS with 3% BSA for 1 h at room temperature, and then incubated overnight at 4 ˚C with either primary anti-Ago2 antibody or blocking solution (control). After washing, the samples were incubated with Alexa-468 anti-rat secondary antibody for 1 hour at room temperature. The slides were then stored at -20 ˚C and imaged using a LEICA TCS SP2 AOBS Spectral Confocal microscope. The images were analyzed and figures were generated using the freeware Vaa3D [73 (link)]. The movie was generated using Volocity® 6.3.