Immunofluorescence Detection of HuR and P-bodies

Cells grown on coverslips were washed twice with PBS containing 10 mM glycine, fixed in 2% paraformaldehyde for 15 min at RT, and permeabilized with 0.02% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 5% normal goat serum in PBS containing 1% IgG-free BSA (Jackson Immunoresearch) and incubated with primary antibody overnight at 4°C diluted in blocking solution. HuR was detected with anti-HuR monoclonal primary antibody (3A2, 1:200 dilution; Santa Cruz Biotechnology) for 1 hr at RT. Secondary antibody incubation was done for 1 hr at RT using FITC-conjugated anti-mouse IgG (1:100 dilution) or Alexa488-conjugated anti-mouse IgG (1:500 dilution; Invitrogen). Cells were counter-stained with DAPI to visualize nuclei. Fluorescence microscopy and image analysis was accomplished as described [31 (link), 61 (link)]. Detection of P-bodies and assessment of P-body co-localization with MS2-YFP was accomplished as described [31 (link)].