P3655 50mg

CSC1589 cells were cultured in 12-well plates at density of 1×10⁵ cells/well and treated with JQ1 for 0.125 or 0.25 µM. Single cell suspensions were seeded on coverslips pre-coated with fibronectin (1 µg/ml, cat. no. F1141-5MG, Sigma-Aldrich; Merck KGaA) and ornithine (50 mg/ml, P3655-50MG, Sigma-Aldrich; Merck KGaA) overnight. CSC1589 cells were fixed with 4% paraformaldehyde at room temperature for 10-15 min and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich; Merck KGaA) in PBS for 5 min. Then, potential non-specific binding sites were blocked with antibody dilution buffer [10% fetal bovine serum and (Sigma-Aldrich; Merck KGaA) and 1% bovine serum albumin (Gentihold) in PBS]. Sections was incubated with an antibody against Nestin (ab6320, 1:50; Abcam) overnight at 4°C. After washing the sections with PBS for 5 min, secondary antibodies were applied [Donkey anti-Mouse IgG (H+L) ReadyProbes™ Secondary Antibody, Alexa Fluor^® 488, R37114, 1:5,000; Invitrogen; Thermo Fisher Scientific, Inc.] for 30 min at room temperature before mounting in water soluble mounting medium (Wuhan Boster Biological Technology, Ltd.) with DAPI (room temperature, 2 min). After the final washing step by PBS, the glass coverslips were mounted upside-down and visualized with an Olympus BX53 microscope at ×400 magnification.