Rabbit anti mad1

Coverslip cultures of fused PtK1 cells were rinsed in PHEM buffer (60 mM 1,4-piperazinediethanesulfonic acid, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM ethylene glycol tetraacetic acid, and 8 mM MgSO₄, pH 7.0), lysed in PHEM buffer supplemented with 1% Triton X-100 for 1 min, and then fixed in 4% paraformaldehyde for 20 min. Cells were rinsed 3 × 5 min in PHEM buffer supplemented with 0.5% Triton X-100 and then blocked for 1 h at room temperature with 10% boiled donkey serum (BDS) in PHEM. Primary antibodies were diluted in 5% BDS in PHEM as follows: mouse anti–α-tubulin, 1:300 (T6199; Sigma-Aldrich) and rabbit anti-Mad1 (1:500; 109519, lot 40030; Gene Tex). Cells were incubated with primary antibodies overnight at 4°C and then rinsed 3 × 5 min in PHEM supplemented with 0.05% Triton X-100. Cells were incubated with appropriate secondary antibodies (donkey anti-mouse or donkey anti-rabbit) conjugated to Alexa Fluor 647 or 555 (Jackson ImmunoResearch Laboratories) diluted 1:300 in 5% BDS in PHEM for 45 min at room temperature. Cells were then rinsed with PHEM supplemented with 0.05% Triton X-100 4 × 5 min, rinsed once with PHEM, and mounted onto slides using mounting medium (20 mM Tris, pH 8.0, 0.5% N-propyl gallate, and 90% glycerol). Coverslips were sealed to the slides using fingernail polish.