Raji Cell Histone Immunocytochemistry and Confocal Help - (Sep/26/2007 )

I am currently working with EBV Raji cells transfected with a Z vector looking at the methylated state of H3. I am currently using a DAR-CY3 antibody for the anti-methyl and a DAM-FITC antibody for the Z staining. As well as staining with Hoechst stain for DNA. My problem is I need to see mitotic chromosomes that are expressing the Z vector and Raji cells are so small its nearly impossible to distinguish mitotic from normal while looking at Z transfected cells. Is there any way to stain for only mitotic chromosomes even though I am already using three dyes, red- anti-methyl, green- anti-Z, and blue- DNA?

It depends on what your microscopy system is capable of. Do you have another filter such as far red? If so you could look at staining for a mitotic marker. I'm not sure if there is a way to specifically stain the mitotic chromosomes. Perhaps there is a specific histone modification that is only found during mitosis. Otherwise you could use something along the lines of cyclin B or Cdk1 phosphorylation to identify which cells are in mitosis. This won't stain the chromosomes but help direct you to the cells you want to analyze. Otherwise, if your system won't allow another stain, you can always isolate the mitotic cells by mitotic shake off. This way the vast majority of cells on your coverslip are mitotic cells.