Rhodamine conjugated goat anti rabbit antibody

Cells grown on coverslips were fixed with 3% paraformaldehyde and permeated with 0.1% Triton X-100. Nonspecific binding was blocked with 1% BSA for 30 min. The cells were then incubated with primary antibodies against rabbit anti-Nogo-B at 4 °C overnight, followed by rhodamine-conjugated goat anti-rabbit antibody (Invitrogen) for 30 min. The cells were then blocked with 1% BSA for 30 min followed by incubating with primary antibodies against rabbit anti-LC3 (Sigma-Aldrich) at 4 °C overnight, followed by rhodamine-conjugated goat anti-rabbit antibody (Invitrogen) for 30 min. Nuclei were counterstained by DAPI (Invitrogen). Images were captured using confocal microscope (Zeiss LSM 700 & Zeiss LSM 880).

KCs were fixed by pre-cooled 4% paraformaldehyde for 30 min, infiltrated by 0.1% Triton X-100 for 10 min, blocked by 1% BSA (Sigma) for 30 min, incubated with primary antibody against F4/80 (Abcam) at 4 ℃ overnight, and maintained with rhodamine-conjugated goat anti-rabbit antibody (Invitrogen) for 30 min, successively. Next, all cells were stained by DAPI (Invitrogen), and photographed using a confocal microscope.

PLC5 cells were plated on glass coverslip and cultured overnight to 30% confluence. Cells were then treated with bufalin or vehicle for 24 hours followed by fixation with 3% paraformaldehyde and permeation with 0.1% Triton X-100. Nonspecific binding sites were blocked with 1% BSA for 30 minutes. The cells were incubated with primary mouse anti-human CCRK antibody (Sigma) or rabbit anti-human β-catenin antibody (Abcam) for 1 hour, and then incubated with FITC-conjugated goat-anti-mouse antibody (Invitrogen) or rhodamine-conjugated goat-anti-rabbit antibody (Invitrogen) for 30 minutes. Nuclei were counterstained by DAPI (Invitrogen) and Images were captured using confocal microscope (Olympus).