Tnf r2 polyclonal antibody

ARPE-19 cells were cultured on glass plates in a cell culture plate (AGC Techno Glass, Shizuoka, Japan) for 24 h, then co-cultured with MT2, TL-Om1 cells using cell culture inserts (Thermo Fisher Scientific, San Jose, CA) for 48 h. ARPE-19 cells were permeabilized and fixed by cold fixing buffer (methanol/acetone, 1:1) at −20°C for 20 min. After blocking with 5% normal goat serum in phosphate-buffered saline for 15 min, cells were incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa Fluor 488-labeled anti-rabbit secondary antibody (Abcam, Cambridge, MA) for 1 h at room temperature. After nuclear staining with 4',6-diamidino-2-phenylindole, cells were scanned using a TCS-SP8 microscope (Leica Micro Systems, Wetzlar, Germany). TNF-R1 polyclonal antibody (Bioss Antibodies, Woburn, MA) and TNF-R2 polyclonal antibody (Proteintech, Chicago, IL), or rabbit IgG control (Abcam) were used as primary antibodies.

The HCT-5 cells were incubated for 10 min in phosphate-buffered saline (PBS) containing 4% paraformaldehyde at 4 °C and immersed in methanol at −20 °C for 10 min. After blocking in 5% normal horse serum in PBS, the cells were incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation in FITC-labeled and tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibodies supplemented with Hoechst dye 33258 for nuclear staining. After being washed in PBS, the cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and scanned using a BIOREVO BZ-9000 fluorescence microscope (Keyence, Tokyo, Japan).
The following antibodies were used as primary antibodies: TNF Receptor 1 Polyclonal Antibody (Bioss Antibodies, Woburn, MA), TNFR2 Polyclonal Antibody (Proteintech, Chicago, IL), and Monoclonal Mouse anti-HTLV-I p19, HTLV-I p28 antibody (Chemicon, Hofheim, Germany).