Mouse anti m13 antibody

For staining of internalized phage particles on live cells, approximately 70,000 HMEC-1 cells were seeded per well in a 4-well permanox chamber slide a day in advance. One hour prior to addition of phage 100 μM chloroquine (Sigma-Aldrich) was added to the media. The cells were moved to room temperature and phage or soluble antibody was added directly to the media and incubated for 30 minutes. The cells were washed one time in fresh medium and moved to 37 °C for 2 hours. After incubation the cells were washed 3 times with EGM and 3 times with PBS and fixed in 2% PFA for 10 minutes at room temperature, residual PFA was removed by PBS. The cells were permeabilized using 0.1% TritonX100 for 15 minutes at RT. The permeabilized cells were blocked 1 hour in 2% mPBS. The cells were incubated 2 hours with mouse anti-M13 antibody (GE Healthcare) (1:4000) in 2% mPBS or anti-Myc-Cy3 (Sigma-Aldrich) (1:1000) For phage staining anti-M13, this was followed by 2 times wash in PBS and incubation with alexa-488 conjugated goat anti-mouse antibody (GE Healthcare) (1:500) in 2% mPBS for minimum 1 hour. The cells were washed 2 times in PBS and mounted using Vectashield mounting media (Vectorlabs).