Alexa fluor 549 conjugated antibodyto rabbit igg

Immunocytochemical analysis was performed as previously described [26 (link)]. KB-VIN cells were grown on an 8-well chamber slide (Lab-Tech) for 24 h prior to treatment with the compound at a concentration of three-fold IC₅₀. CA-4 was used at 0.2 μM (Figure 2A and Supporting Information, Supplementary Figure S1). After treatment of cells with the agent for 24 h, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were labeled with mouse monoclonal antibody to α-tubulin (B5-1-2, Sigma) and rabbit IgG to Ser10-phosphorylated histone H3 (p-H3) (#06570, EMD Millipore), followed by FITC-conjugated antibody to mouse IgG (Sigma) and Alexa Fluor 549-conjugated antibody to rabbit IgG (Life Technologies). Nuclei were labeled with DAPI (Sigma). Fluorescently-labeled cells were observed using a confocal microscope (Zeiss, LSM700) with ZEN (black edition) software (Zeiss). The 15~20 optical sections acquired at 0.5~1 µm intervals were stacked and reconstructed using ZEN (black edition) software. Experiments were repeated at least twice for each compound. Final images were prepared using Adobe Photoshop CS3.