Alexa flour 568 goat anti rabbit igg

For the colocalization studies, THK cells (4 × 10⁴ cells per well) were seeded onto glass coverslips in 24-well culture plates, as mentioned above; this was followed by co-transfection with OnSARM1-3×FLAG and HA-tagged OnTRIF, OnTIRAP (27 (link)) or OnMyD88 (21 (link)) expression plasmids, respectively. The cells were fixed 48 h after transfection; this was followed by permeabilizing with PBS containing 1% (v/v) triton-X100 at room temperature for 5 min and blocking with PBS containing 10% bovine serum albumin (BSA) for 30 min at 37°C. Subsequently, the cells were incubated with PBS containing 3% BSA and monoclonal anti-FLAG M2 mouse antibodies (1:200, Sigma-Aldrich) or anti-HA rabbit antibodies (1:200, clone C29F4, Cell Signaling) for 2 h at 37°C. Alexa Flour™ 488 goat anti-mouse IgG (1:1000, Invitrogen) and Alexa Flour™ 568 goat Anti-rabbit IgG (1:1000, Invitrogen) were used in dark to visualize FLAG tagged (green) or HA tagged proteins (red), respectively. The cells were then stained with VECTASHIELD Antifade Mounting Medium containing DAPI to visualize the nucleus (blue). The images were captured using confocal microscopy (Zeiss LSM 880 with Airyscan). The colocalization efficiency was quantified by observing the overlap of the fluorescence intensity peaks along the white line by line scan analysis profiles (ZEISS ZEN 2.6 (blue edition) software).