Ab61195

The expression levels of Foxp3 and CD25 receptor protein were examined immunohistochemically in pericardial fluid biopsy samples obtained from the patients with CHF and NHF. Staining of CD25 (1:100; ab61195, Abcam, Cambridge, MA, USA) and Foxp3 (1:100; sc-28705, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) immunohistochemistry was performed on formalin-fixed sections, followed by incubation with PV-6001 (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. Each stained histological section was examined under an Olympus BX53F microscope (Olympus Corporation) connected to a computerized image-analysis system (Image-Pro v6.0; Media Cybernetics Inc., Silver Spring, MD, USA).

The samples were lysed in RIPA lysis buffer with protease inhibitor cocktail (Beyotime Biotechnology, Beijing, China) for 30 min on ice. The proteins were separated by SDS-PAGE and then transferred onto 0.45 μm NC membranes (Millipore) at 300 mA for 2 h. The membranes were blocked with 5% non-fat milk and then incubated with primary antibodies. Antibodies used were: GAPDH (60004-1-Ig, Proteintech), SOX4 (ab236557, Abcam), NRP1 (PA1-29513, Invitrogen™), UNG (PA5-20265, Invitrogen™), IVNS1ABP (ab127566, Abcam), and IL2RB (ab61195, Abcam). The membranes were then incubated with appropriate HRP-conjugated secondary anti-rabbit IgG antibodies for 1 h at room temperature after washed four times with TBST. The results were visualized by the ChemiDoc Imaging System (Bio-Rad, American).