Alexa fluor 488 goat anti rabbit immunoglobulin igg

Tissues were fixed in 10% formalin in OCT compound (Leica, Buffalo Grove, IL, USA) for the analysis of the localization of Prx3 in mitochondria. The samples were sectioned at a thickness of 7 μm. The tissues were blocked using blocking solution (DAKO) for 1 h in the dark. The primary antibody against Prx3 (1:100; Abcam) was added to the diluent solution (DAKO) and incubated with sections at 4 °C overnight. The secondary antibody, Alexa Fluor 488 goat anti-rabbit immunoglobulin IgG (Invitrogen), was then added for 1 h. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). The images were observed with a confocal microscope (Zeiss, Oberkochen, Germany). These experiments were performed in triplicate.

ARPE-19 cells were seeded on 8-well cell culture plates. After treatment with 40 μM of luteolin or DMSO as control, the cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 5 min at room temperature, and blocked with 1% bovine serum albumin in PBS for 1 h. The fixed cells were incubated with primary antibodies against ZO-1 and vimentin at 4°C overnight, then washed with PBS, and incubated with the appropriate secondary antibody (Alexa Fluor® 488 goat anti-rabbit immunoglobulin IgG, Invitrogen Corporation) followed by DNA staining with DAPI for 5 min at room temperature. Then, the cell culture plates were mounted on glass slides and observed under a laser confocal microscope (Zeiss LSM880, Carl Zeiss, Oberkochen, Germany) equipped with ZEN image processing software.