Secondary fluorescent conjugated antibodies

Immunocytochemistry was performed to target several different antibodies (see supplementary material Table S2). For immunostaining, cells were grown in vitro on glass coverslips pre-treated with poly-L-Lysine (Sigma), Laminin (Sigma) and Matrigel (BD Biosciences). Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stored in PBS at 4°C short term. Cells were permeabilized using 0.1% Triton X-100 in PBS for 1 h at room temperature. Blocking was performed by adding 5% Normal Donkey Serum (Sigma) in PBS (blocking buffer) to the cells and cultured for 1 h at room temperature. Immunolabeling with the primary antibody was performed overnight. Antibodies used and their dilutions are listed in supplementary material Table S2. Cells were rinsed three times using PBS and cultured in secondary fluorescent-conjugated antibodies (Jackson ImmunoResearch) at 1:200 dilution in blocking buffer. Cells were labeled with Hoechst (5 µg/ml). As a negative control, cells were cultured in the absence of the primary antibody and also with a matching isotype instead of the primary antibody. Cells were visualized using a DMRB fluorescent microscope (Leica Microsystems) and a TCS SPE confocal microscope (Leica Microsystems). Images were captured using Leica Application suite (2.8.1) capture and MM-AF software (Leica Microsystems).

Primary antibody anti-α-tubulin (Sigma:
T9026) was utilized. All appropriate secondary fluorescent conjugated
antibodies were purchased from Jackson ImmunoResearch.