Visualizing Protein Expression in Cells

Expression and distribution of various proteins were visualized by indirect immunofluorescence. Cells were fixed for 10 min in PBS/4% (w/v) paraformaldehyde, and permeabilized for 7 min using PBS/0.2% (v/v) Triton X-100. Non-specific sites were blocked with PBS/10% goat serum (1 hr, room temperature [RT]) prior to incubation with antibodies diluted in PBS/5% goat serum (1 hr, RT, each). F-actin was detected by incubating cells with TRITC-phalloidin (Sigma) for 1 hr at RT, and nuclei were stained using 4 μg/mL Hoechst 33,258 (Sigma) for 5 min at RT. Cells were washed in PBS before mounting in ProLong Gold Antifade (Molecular Probes). Images were collected on a Nikon A1 confocal or a Leica TCS SP5 AOBS inverted confocal microscope as previously described (Akhtar and Streuli, 2013 (link)). Non-biased cell counts were performed by concealing the identity of each slide.
Immunofluorescence of mammary tissue was performed on paraffin-embedded tissue (5 μm) or cryosections (10 μm), and the luminal surface was detected with wheat germ agglutinin-488, or -647 (Invitrogen, W11261, W32466) and imaged using confocal microscopy. Primary antibodies used for immunofluorescence are indicated in Supplemental Experimental Procedures. Secondary antibodies conjugated to Cy2, Rhodamine-RX, and Cy5 (Jackson ImmunoResearch).