Murine bone marrow cells were collected from femurs (see later section in Methods) and treated with colcemid (6 ml/mL in 0.075 M KCl) for 2 hours. The cells were fixed using Carnoy's fixative (3:1; methanol:acetic acid), dropped onto non-silanized slides, and air-dried. The slides were baked at 60uC for 12 hours and aged at room temperature for 72 hours. Prior to banding cells were aged at 90uC for 15 minutes. Metaphases were GTG banded and photographed with the BandView imaging system from Applied Spectral Imaging (ASI). Upon completion of the GTG analysis, the slide was de-oiled in Americlear. Slides were de-stained in methanol for 5 minutes and rehydrated in an ethanol series: 100%, 80%, and 70% ethanol. Subsequently, the slides were post-fixed in 1% formaldehyde/1XPBS/50mM MgCl 2 for 2 minutes at room temperature, washed in 1x PBS for 5 minutes and dehydrated in 70%, 80%, and 100% ethanol series. Once the slides were airdried, they were denatured in 70% formamide/2x SSC at 72uC for 30 seconds and immediately placed in cold 70%, 80%, and 100% ethanol and air-dried. Denatured mouse SkyPaint probe (10 ml, Applied Spectral Imaging) was applied to each slide, covered with a glass coverslip and sealed with rubber cement. Slides were transferred to a humidified chamber and placed in a 37uC incubator for 48 hours. Post-washes and detection was done following ASI SKY protocol. SKY metaphases were captured and analyzed with the SkyVision spectral imaging system (Applied Spectral Imaging). CEACAM1 BAC2 DNA was used as a probe. Three nick reactions were prepared using digoxigenin-dUTP. Reactions were combined for precipitation and re-suspended in 10 ml of 10 mM Tris buffer pH 8.5 for hybridization. The probe was hybridized to a human male metaphase to check for probe intensity and correct integration site. The probe (100 ng) was combined with 9 mL hybridization buffer, co-denatured on an 80u hotplate for 5 minutes and incubated overnight at 37uC in a humidified chamber. Post-wash consisted of 50% formamide/2x SSC at 45uC for 15 minutes, 2x SSC at 37uC for 8 minutes and 1x PBD at room temperature for 2 minutes. Probe was detected with rhodamine anti-digoxigenin and following SKY imaging, the slide was placed in 1x PBD for coverslip removal and immersed in room temperature 2x SSC for 1 hour to remove the SKY probe. After SSC treatment, slides were dehydrated and co-denatured for 3 minutes. Hybridization and post-wash were run as described above. Bone marrow preparations from a wild type mouse were used as a negative control and normal human male preparations were used as a positive control. Total murine bone marrow cells were prepared as described in a later section in Methods. Male bone marrow cells were discard tissue from the City of Hope bone marrow transplantation unit.