Alexa 488 conjugated anti rat igg

All sections were deparaffinized, and antigen retrieval was performed as previously described. For the detection of M2-like TAMs, lymph nodes or lung tissues were blocked with 1.5% BSA containing 0.2% Triton X-100. The tissues were incubated with rat anti-mouse CD206 primary antibodies (1:1000; Bio-Rad Laboratories) and visualized using Alexa-488 conjugated anti-rat IgG (1:1000; Invitrogen). All antibodies were diluted in 0.5% BSA solution. To detect TAMs and CD8⁺ T cells in tumor nodules of lung tissues, the sections were incubated overnight at 4 °C with the following antibodies: rabbit anti-mouse CD8a (1:2000; Abcam, Cambridge, MA, USA) and rat anti-mouse CD68 (1:1000; Bio-Rad Laboratories). They were then visualized after incubating with the following antibodies for 1 h at room temperature: Alexa-594 conjugated anti-rabbit IgG (1:1000; Invitrogen) and Alexa-488 conjugated anti-rat IgG (1:1000; Invitrogen). Five fields of different tumor nodules in the lungs were randomly selected and visualized using an LSM 800 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

After the real-time luciferase assay, the slices were fixed for several hours with 4% paraformaldehyde in 0.1 M PBS. The slices were incubated at 4°C overnight with rabbit anti-Cux1 (1:250; Santa Cruz Biotechnology) and rat anti-GFP (1:1,000; nacalai tesque). After extensive washes, the signals were visualized with Alexa 488-conjugated anti-rat IgG (1:500; Invitrogen) and Cy5-conjugated anti-rabbit IgG (1:250; Jackson ImmunoResearch). The samples were embedded with 80% glycerol containing DAPI and DABCO, and observed by confocal microscopy through a 20× objective lens (Leica, TCS-SP5).

Anti-Aurora B (ab2254), anti-INCENP (ab36453) and anti-HA (16B12; ab130275) antibodies were obtained from Abcam. Anti-Plk pT210 (sc-135706) and normal rabbit IgG were obtained from Santa Cruz Biotechnology. Anti-Cdc7 antibody was obtained from MBL. Anti-Aurora B pT232 antibody (Poly6361) obtained from BioLegend was used only for immunostaining. Anti-Histone H3 pS10 rabbit antibody was from Upstate. Anti-Histone H3 pS28 antibody was prepared in house in rat^{70 (link)}. Anti-CENP-A pSer7 antibody (2187) was from Cell Signaling Technology. Antibodies against Tubulin, Aurora-A and FLAG (M2) were obtained from Sigma. Alexa 488 conjugated anti-rabbit IgG, Alexa 546 conjugated anti-rabbit IgG, Alexa 488 conjugated anti-rat IgG, and Alexa 647 conjugated anti-rabbit IgG, obtained from Invitrogen, were used for immune-staining or FACS staining.