Alexa fluor 594 conjugated anti mouse igg

CKO cells (∼6 × 10⁵) were plated on glass coverslips in 12-well plates prior to transfection with 0.5–1 μg pLB(N)CX-CPSF6[551] or CPSF6 mutant expressing derivative using Effectene or PolyJet. At 24 h post-transfection, cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, washed three times with PBS, permeabilized with 0.5% triton X-100 for 15 min at room temperature, and rewashed three times with PBS. The cells were then blocked by adding 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, followed by staining with rabbit monoclonal anti-CPSF6 antibody and mouse monoclonal anti-α-tubulin antibody (Santa Cruz). After overnight incubation at 4°C, cells were washed three times with PBST, and then stained with secondary Alexa Fluor 488 conjugated-anti-rabbit IgG (Abcam) and Alexa Fluor 594 conjugated-anti-mouse IgG (Abcam) antibodies (see Supplementary Table S3) for 1 h at room temperature. After rinsing three times with PBST, the cells were covered with mounting Prolong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) and analyzed using a 100x objective lens under oil with lasers (405 nm, DAPI; 488 nm, Alexa Fluor 488; 561 nm, Alexa Fluor 594) on a Yokogawa spinning disk confocal microscope at the Dana-Farber Cancer Institute (DFCI) Confocal and Light Microscopy Core Facility.