Immunofluorescence was performed as previously described (Hicks et al., 2015 (link)). In brief, cells were fixed in 4% formaldehyde (Pierce, ThermoFisher) for 5 min at room temperature. The cells were permeabilized by washing in PBST (PBS +0.1% Triton X-100). The primary antibody was incubated in blocking buffer (PBS +5% Normal goat serum) overnight at 4°C. Next, the samples were washed three times in PBST and incubated for 1 h at room temperature with the fluorescent secondary antibody (Goat anti-mouse or Goat anti-rabbit antibodies conjugated with either Alexa488 or Alexa555, 1:400 dilution, Life technologies) in the same blocking buffer. The samples were then washed four times with PBST. In order to visualize nuclei, the cells were stained with DAPI. For visualizing the actin cytoskeleton Alexa633 conjugated Phalloidin was used (Life technologies; 1:400). For detecting biotinylated proteins, the samples were incubated with Streptavidin Alexa647 (Life technologies; 1:1,200). The Strep674 was added to the sample at the same time as the secondary antibody. The cells were mounted onto slides using Prolong Diamond (Life technologies).
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