Rabbit anti cd45

Cells on slides were permeabilized with 0.2% Triton X-100 in PBS for 10 min and quenched with 0.3% hydrogen peroxide for 30 min at room temperature. Cells were then blocked with 1% bovine serum albumin in PBS for 30 min and incubated with primary antibodies for 90 min at 37°C, followed by secondary antibody incubation at same condition. The primary antibodies used were mouse anti-EpCAM (dilution, 1:200; catalog no. #2929; CST Biological Reagents Company Ltd., Shanghai, China) and rabbit anti-CD45 (dilution, 1:50; catalog no. SC53047; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). EpCAM signaling was amplified using the TSA™ kit (catalog no. T20922; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The secondary antibody used against CD45 was goat anti-rabbit Alexa Fluor^® 594 (dilution, 1:100; catalog no. A11012; Invitrogen; Thermo Fisher Scientific, Inc.). The slides were mounted using Fluoroshield™ Mounting Medium with DAPI (ImmunoBioScience Corp., Mukilteo, WA, USA). Stained cells were observed and images captured using a fluorescent microscope (Eclipse Ti; Nikon Corporation, Tokyo, Japan) with a 400X objective. To identify EpCAM-positive and CD45-negative CTCs, the PC9 (EpCAM-positive) and KG-1 (CD45-positive) cell lines were used as positive controls.