Goat anti rabbit conjugated with alexafluor 633

Cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT and permeabilized in methanol for 5 min at −20 °C. Cells were then blocked with PBS containing 4% BSA for 30 min and incubated with the following primary antibody diluted in the blocking solution overnight at 4 °C: rabbit acetylated α-tubulin (1:4000; Cell Signaling Technology, Inc., Danvers, MA, USA), as previously described^{22 (link),33} . Cells were then rinsed in PBS several times before incubation with secondary antibodies: goat anti-rabbit conjugated with Alexafluor 633 (1:2000; Life Technologies, CA, USA) for 30 min at RT. After extensive washing, coverslips were mounted with Vectashield mounting medium (Vector Laboratories Burlingame, CA, USA) with DAPI and then observed at a Leica TCS SP5 confocal microscope (Leica).

Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature (RT) and permeabilized in methanol for 5 min at -20°C. Cells were then blocked with PBS containing 4% BSA for 30 min and incubated with the following primary antibodies diluted in the blocking solution overnight at 4°C: rabbit ß-tubulin (Abcam, Cambridge, UK ) 1:200; mouse α-tubulin (Abcam) 1:1000; mouse acetylated α-tubulin (Abcam) 1:500. Cells were then rinsed in PBS several times before incubation with secondary antibodies, goat anti rabbit conjugated with Alexafluor 633 (1:2000), goat anti mouse conjugated with Alexafluor 488 (1:2000), (Life Technologies, CA, USA) for 30 min at RT. After extensive washing, coverslips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories Burlingame, CA, USA) and then observed at a Leica TCS SP5 confocal microscope (Mannheim, Germany).