FISH experiments were performed as previously described54 (link),55 (link). To generate FISH probes, plasmid, cosmid, or PCR products were labeled by incorporation of Cy3-dCTP or Cy5-dCTP (GE Healthcare) with a random primer DNA labeling kit (Takara). The plasmid pRS140 and cosmid cos212 were used to prepare FISH probes against centromeres and telomeres, respectively54 (link),56 (link). FISH probes for other gene loci were generated using PCR-amplified DNA fragments (~15 kb).
IF experiments were performed as previously described52 (link),57 (link). Fixed cells were incubated with primary antibodies such as 1:10,000 diluted rabbit polyclonal anti-Myc (ab9106, Abcam) and 1:1,000 diluted mouse monoclonal anti-Pk (SV5-Pk1, Serotech). Cells were subsequently incubated with secondary antibodies such as 1:1,000 diluted Cy3-conjugated anti-mouse IgG (115-165-003, Jackson ImmunoResearch) and 1:1,000 diluted Alexa Flour 488-conjugated anti-rabbit IgG (A11034, Molecular Probes). FISH and IF images were captured using a Zeiss Axioimager Z1 fluorescence microscope with an oil immersion objective lens (Plan Apochromat, 100×, NA 1.4, Zeiss). The images were acquired at 0.2-μm intervals in the z axis controlled by Axiovision 4.6.3 software (Zeiss). More than 100 cells were analyzed for microscopic experiments.
IF experiments were performed as previously described52 (link),57 (link). Fixed cells were incubated with primary antibodies such as 1:10,000 diluted rabbit polyclonal anti-Myc (ab9106, Abcam) and 1:1,000 diluted mouse monoclonal anti-Pk (SV5-Pk1, Serotech). Cells were subsequently incubated with secondary antibodies such as 1:1,000 diluted Cy3-conjugated anti-mouse IgG (115-165-003, Jackson ImmunoResearch) and 1:1,000 diluted Alexa Flour 488-conjugated anti-rabbit IgG (A11034, Molecular Probes). FISH and IF images were captured using a Zeiss Axioimager Z1 fluorescence microscope with an oil immersion objective lens (Plan Apochromat, 100×, NA 1.4, Zeiss). The images were acquired at 0.2-μm intervals in the z axis controlled by Axiovision 4.6.3 software (Zeiss). More than 100 cells were analyzed for microscopic experiments.
