Immunofluorescence Staining of Cytoskeletal Proteins

Cells were grown on glass coverslips and fixed with 3.7% formaldehyde in PBS (containing Ca²⁺ and Mg²⁺) for 10 min followed by permeabilization for 10 min with 0.1% Triton X-100 in PBS (Ca²⁺ and Mg²⁺). Cells were blocked in PBS (Ca²⁺ and Mg²⁺) containing 10% FBS and labelled with primary antibodies as indicated. Cells were washed 3 times followed by labelling with Alexa Fluor secondary antibodies. Staining of F-actin was carried out using Phalloidin-568 (Molecular Probes). DNA staining was performed with 4′,6-Diamidino-2-phenylindole (DAPI [Molecular Probes]) after which the coverslips were mounted in Vectashield solution. For endogenous KIF4 and PRC1 stainings, cells were pre-permeabilized for 3–5 min in PHEM buffer (100 mM PIPES pH 6.8, 25 mM HEPES pH 7.4, 5 mM EGTA, 2.5 mM MgCl₂) plus 0.5% Triton X-100. Then, cells were fixed for 10 min in PHEM buffer containing 3.7% formaldehyde. Mitotic PRC1 phosphorylation was monitored after ice-cold methanol fixation. Calcium-stable microtubule stainings were carried out as described previously50 (link). Subsequent steps were performed as described above. z-stacks with 0.2 μm spacing were acquired using a DeltaVision Elite system (Applied Precision). Maximum intensity projection of the z-levels were analysed with ImageJ (National Institute of Health) and processed using Photoshop and Illustrator software (Adobe).