Goat anti rabbit cy3

Huh-7, Bel-7402, SNU387, and SNU449 cells were seeded onto glass slides. At 48 h after transfection with the NAT10 siRNA or Twist siRNA or treatment with remodelin in the presence of doxorubicin or hypoxia, the cells were rinsed with PBS, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked for 30 min in 10% BSA, and then incubated with an anti-E-cadherin monoclonal antibody (1 : 200; Cell Signaling Technology) or anti-vimentin monoclonal antibody (1 : 200; Cell Signaling Technology) overnight at 4°C. After three washes in PBS, the slides were incubated with goat anti-rabbit Cy3 as a secondary antibody (1 : 200; Cell Signaling Technology) for 1 h in the dark. After three further washes, the cells were stained with DAPI for 5 min to visualize nuclei and examined by confocal microscopy (Olympus, Tokyo, Japan).

For the detection of Tnfα and Tgfβ1 2 days after lesion with 6-OHDA, sections from SN and CPu were washed with PBS and blocked with PBS containing 10% normal goat serum and 0.1% TritonX-100 (Roche) for 1 h at RT. Afterwards, sections were incubated with primary antibodies anti-Tnfα (sc-52746, 1:100, Santa Cruz), anti-Tgfβ1 (MAB240, R&D System, 1:50, Wiesbaden-Nordenstedt) and anti-Iba1 (1:500, Wako Chemicals) at 4°C overnight, followed by an incubation with the corresponding Cy3-conjugated secondary antibodies (goat anti-mouse Cy3 1:100, goat anti-rabbit Cy3 1:200, Cell Signaling Technologies) for 1 h at RT. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, Roche). Fluorescence images were captured using the ZEISS AxioImager M2 imaging system (ZEISS, Göttingen, Germany).