Alexa fluor 568 conjugated anti rabbit igg antibody

Immunofluorescence staining of cells was performed in chambered slides (BD Bioscience, San Jose, CA, USA). Cells were seeded in chambered slides and fixed in ice-cold methanol for 10 min. Slides were then washed in phosphate-buffered saline (PBS) three times, and they were incubated with blocking solution (3% BSA in PBS) for 30 min. For immunofluorescence staining of mouse tissues, formalin-fixed, paraffin-embedded sections were used. Slides were deparaffinized and dehydrated. Mouse monoclonal acetylated α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) and non-phospho β-catenin antibody (Cell signaling, Danvers, MA, USA) was used as primary antibodies. Samples were then stained with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Life technologies, Carlsbad, CA, USA) and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA, USA) with DAPI (4′,6-diamidino-2-phenylindole).

Antibodies against β-actin, FLAG, GFP (Sigma-Aldrich), CDC20, phosphor-histone H3^Ser10 (Santa Cruz Biotechnology), phosphor-CDK1^Tyr15, CDC27, CHK2 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), CDH1 (Thermo Fisher Scientific, Waltham, MA, USA), phosphor-CHK2^Thr68 (Calbiochem, San Diego, CA, USA), and ENSA (also recognize ARPP19) (Abcam, Cambridge, UK) were obtained from the indicated suppliers. Antibodies against CDK1 and cyclin B1 were gifts from Julian Gannon (Cancer Research UK). Rabbit polyclonal antibodies against MASTL were raised against bacterially expressed GST-MASTL^C∆310 and then purified with a method involving membranes loaded with purified GST-MASTL^C∆31028 (link). Immunoblotting and immunoprecipitation were performed as described previously25 (link). For immunostaining of 53BP1, cells were washed twice with ice-cold PBS and fixed with methanol at −20 °C for 10 min followed by blocking with 3% BSA in 0.2% Tween-20 in PBS at 25 °C for 30 min. The cells were then incubated with anti-53BP1 antibody (H-300; Santa Cruz Biotechnology) at 4 °C for overnight. The samples were then incubated with Alexa Fluor 568 conjugated anti-rabbit IgG antibody (Life Technologies) at 25 °C for 2 h. The cells were counterstained with Hoechst 33342 for 1 min and viewed under fluorescence microscope using a 40X objective.