Alexa fluor488 labeled anti rabbit secondary antibody

ARPE-19 cells were cultured on glass bottom plates (AGC Techno Glass, Shizuoka, Japan) for 24 h, then co-cultured with MT2 and Jurkat cells using cell culture inserts (Thermo Fisher Scientific) for 48 h. After three washes with phosphate-buffered saline (PBS) (Wako Pure Chemical Corporation), ARPE-19 cells were fixed by cold fixing buffer (methanol/acetone, 1:1) at −20°C for 20 min and blocked with 10% FBS in PBS for 15 min. Cells were then incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa fluor488-labeled anti-rabbit secondary antibody (Abcam, Tokyo, Japan) along with 4′,6-diamidino-2-phenylindole dihydrochloride (Cosmo Bio, Tokyo, Japan) incubation for 1 h at room temperature. The following antibodies were used as primary antibodies: TNF receptor 1 polyclonal antibody (Bioss Antibodies, Woburn, MA) and TNF receptor 2 polyclonal antibody (Proteintech, Chicago, IL). We scanned using a TCS-SP8 microscope (Leica Micro Systems, Wetzlar, Germany).

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.