HeLa cells were fixed with 100% methanol for EB1 immunostaining and PLA experiments (anti-EB1 antibody). All other immunostaining and PLA experiments were performed after cofixing cells with 4% paraformaldehyde, PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 4 mM MgCl2, pH 6.9), and 0.5% Triton X-100 for 20 min. Immunofluorescence staining was performed in 3% BSA/TBS–0.05% Tween 20 using these antibodies anti-tubulin DM1-α, antiphospho-H3Ser10 (EMD Millipore), antiphospho–histone H3T3 (EMD Millipore), antiphospho-H2AT120 (Active Motif), phospho-KNL1 (S24 and S60; I.M. Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, MA; Welburn et al., 2010 (link)), anti-AIM1 (Aurora B), anticentromere antigen (Antibodies, Inc.), anti-hEB1, and TO-PRO-3 (Invitrogen).
After the PLA procedure, cells were incubated in 10% normal mouse serum and 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc.) in 3% BSA/TBS + 0.05% Tween 20 for 30 min at RT to block any open binding sites on the PLA probes. FITC-conjugated anti–α-tubulin, DM1-α, and Alexa Fluor 647–conjugated polyclonal antibodies (xlNdc80, xlINCENP, or hBorealin) were used for costaining with PLA reactions. Alexa Fluor 647 polyclonal antibody conjugations were prepared using an Alexa Fluor 647 labeling kit following the manufacturer’s recommended protocol (Invitrogen).
After the PLA procedure, cells were incubated in 10% normal mouse serum and 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc.) in 3% BSA/TBS + 0.05% Tween 20 for 30 min at RT to block any open binding sites on the PLA probes. FITC-conjugated anti–α-tubulin, DM1-α, and Alexa Fluor 647–conjugated polyclonal antibodies (xlNdc80, xlINCENP, or hBorealin) were used for costaining with PLA reactions. Alexa Fluor 647 polyclonal antibody conjugations were prepared using an Alexa Fluor 647 labeling kit following the manufacturer’s recommended protocol (Invitrogen).
