Mouse anti p150 glued

For immunofluorescence, cells were plated on #1.5 25 mm coverslips coated with 1 mg/mL poly-L-lysine. Cells were fixed with 95% methanol + 5 mM EGTA at −20°C for 3 min, washed with TBS-T (0.1% Triton-X-100 in TBS), and blocked with 2% BSA in TBS-T for 1 hr. Primary and secondary antibodies were diluted in TBS-T+2% BSA and incubated with cells overnight at 4°C (primary) or for 20 min at room temperature (secondary). DNA was labeled with Hoescht 33342 (Sigma, St. Louis, MO) before cells were mounted in ProLongGold Antifade (P36934; Thermo Fisher). Cells were imaged using the spinning disk confocal microscope described above. Antibodies: mouse anti-α-tubulin DM1α (T6199; Sigma), rabbit anti-α-tubulin (ab18251; Abcam, Cambridge, UK), rabbit anti-NuMA (NB500-174; Novus Biologicals, Littleton, CO), mouse anti-p150-Glued (610473; BD Biosciences, San Jose, CA), mouse anti-α-tubulin DM1α conjugated to Alexa488 (8058S; Cell Signaling, Danvers, MA), mouse anti-dynein intermediate chain (MAB1618MI; Millipore, Billerica, MA), rabbit anti-EB1 (sc-15347; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-KANSL1 (PAB20355; Abnova, Taipei City, Taiwan), rabbit anti-CAMSAP1 (NBP1-26645; Novus Biologicals), mouse anti-actin (MAB1501; Millipore), rabbit anti-γ-tubulin (T3559; Sigma), and camel nanobody against GFP coupled to Atto488 (gba-488; ChromoTek, Hauppauge, NY).