Ab109524

Cells were grown on glass coverslips, washed with phosphate-buffered saline (PBS), fixed with 1 or 4% paraformaldehyde (in PBS) for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After blocking with 2% bovine serum albumin (BSA) in PBS for at least 1 h, cells were incubated with primary antibodies, diluted in 2% BSA in PBS for 1 h at room temperature (RT) or overnight at 4°C, washed three times with PBS, and incubated with secondary antibodies in 2% BSA in PBS for 1 h at RT. After immunostaining, cells were washed with PBS and mounted on microscopic slides with VectaShield (VectorLabs) for microscopic analysis. The following primary antibodies were used in this study: anti-calnexin (Abcam, ab22595), anti-calreticulin (ThermoScientific, PA3-900), CREST (ImmunoVision, HCT0100), anti–Ki-67 (Abcam, ab16667), anti-KIF4 (Abcam, ab3815), anti-KIF22 (Abcam, ab222187), anti-LBR (abcam, ab32535), mAb414 (Abcam, ab24609), anti-MAD2 (Bethyl Laboratories, A300-301A), anti-topoisomeraseII (Abcam, ab109524), anti–α-tubulin (Sigma, T5168), and anti–γ-H2AX (Millipore, 05-636-l). The antibody against human SUN1 has been previously described (Sosa et al., 2013 (link)). For secondary antibodies, Alexa Fluor–conjugated antibodies (goat, 1:300; Life Technologies) were used.