Donkey anti mouse coupled to alexa fluor 488

BHK cells were grown on 24-well tissue-culture plates with glass coverslips. Permeabilized cells were fixed with 4% paraformaldehyde (EM grade, Bar Naor, Israel) in PBS, followed by incubation in 40 mM NH₄Cl to block free aldehydes, permeabilized in 0.1% Triton X-100 in PBS and blocked in 1% FBS in PBS. After fixation, the coverslips were incubated 1 h with mouse anti-V5 antibody (Invitrogen, 1:500) and 1 h with the secondary antibody which was donkey anti-mouse coupled to Alexa Fluor 488 (Invitrogen, 1:500). Alternatively, for immunofluorescence without permeabilization, cells were blocked on ice in PBS with 1% FBS for 20 min, and then stained with Monoclonal ANTI-FLAG M2 antibody (Sigma, 1:1000) on ice for 1 h. After anti-FLAG staining, cells were washed and fixed with 4% PFA in PBS. Cells were blocked again and stained with the secondary antibody (donkey anti-mouse coupled to Alexa Fluor 488; Invitrogen) diluted 1:500 in PBS for 1 h. In all cases, nuclei were stained with 1 µg/ml DAPI. Images were captured using a Nikon Eclipse E800 with a 60X/1.40 Plan Apochromat objective and an optical zoom lens (Nikon) using a Hamamatsu ORCA-ER camera controlled by Micro-Manager software^{122 (link)} (Fig. 5f).