Results of DISH before and after individualized optimization for a sample from year 2003. (A) U3 protocol was used for first DISH analysis. There were no red and black signals within the nuclei, and there was SISH dust in the background. As the nuclear morphology was preserved, further optimization could be done to optimize the staining process. (B) U6 protocol was used for the second DISH analysis and was successful. There were enumerable red and black signals within the nuclei after a longer duration of cell conditioning with CC2 and longer ISH protease 2 treatments were used. The lengths of incubation for SISH and Red ISH multimers, silver and red ISH chromogens, Hematoxylin II counterstain and bluing reagent were decreased so as to reduce unspecific background staining (SISH dust) and intensity of counterstain. Original magnification (A, B: 600×).