Af594

An ~800 bp purified P2A-mCherry DNA fragment was labeled with a red-dUTP (AF594, Molecular Probes) by nick translation, and a HOXA9 BAC clone (CH17-412I12/7p15.2) was labeled with a green-dUTP (AF488, Molecular Probes). Both of labeled probes were combined with sheared human DNA and independently hybridized to fix the interphase and metaphase nuclei derived from each sample by using routine cytogenetic methods in a solution containing 50% formamide, 10% dextran sulfate, and 2XSSC. The cells were then stained with DAPI and analyzed.

The Cancer Center Core Cytogenetic Laboratory received 10 formalin fixed paraffin embedded (FFPE) retinoblastoma tissue specimens in order to determine by FISH if there is a disruption of the RB1 gene in these tumors. Purified BAC DNA from two RB1 3-prime clones (RPH-115122 and RP11-90K7) were labeled with a green-dUTP (AF488, Molecular Probes) by nick translation, and one RB1 5-prime clone (RP11-795F23) was labeled with a red-dUTP (AF 594, Molecular Probes). In normal cells that contain normal RB1 gene this probe set produces very tightly linked red and green signals since the probes are separated by only 80kb. This assay was specifically designed to detect any disruption of the RB1 gene occurring between introns 6 and 16. One hundred interphase nuclei from each tumor were scored for the presence of either normal RB1 genes (tightly linked red and green signals) or disrupted or deleted RB1 genes (separated red and green signals or deletion of either one). Details for FISH protocol have previously been described [8 (link)].

Purified human NMYC BAC DNA (RP11-1183P10) was labeled with a red-dUTP (AF594, Molecular Probes) and human chromosome 2 control (2q11.2) BAC DNA (RP11-527J8) was labeled with a green-dUTP (AF488, Molecular Probes) both by nick translation. The paraffin slides were deparaffinized with xylene 2 × 10 min each at room temperature (RT), placed in ethyl alcohol 3 × 2 min each at RT, air-dried, placed in 10% buffered formalin for 1 h at RT. The slides were then placed in pepsin (8 mg/ml) in 0.1 n HCL for 3 min, rinsed in dH₂O for 5 min at RT. One-hour incubation in Carnoy’s fixative at RT was performed. The labeled probes were combined with human sheared DNA and hybridized to the slides in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC. The probe and slide were co-denatured at 90 °C for 12 min and incubated overnight at 37 °C on a Thermobrite. In brief, washed in PN and then stained with 4,6-diamidino-2- phenylindole (DAPI) (1 µg/ml). Images were captured using a Plan-Apochromat × 63 objective on a Zeiss Axio Imager.Z2 microscope and GenASIs scanner (ScanView software version 7.2.7).

Cells were grown to 60–70% confluence, treated with colcemid (0.02 µg/mL) for a 4 h incubation at 37 °C, and harvested using routine cytogenetic methods. The cells were then trypsinized for 5 minutes at 37 °C and centrifuged at 900 RPM for 5 minutes. The cell pellet was resuspended in 0.075 M KCL for 8 minutes at RT and then centrifuged at 600 RPM for 5 minutes, resuspended in 3:1 Carnoy’s fixative (3 parts methanol: 1 part acetic acid), and incubated for 15 minutes at RT. The cells were centrifuged at 600 RPM for 5 minutes, resuspended in Carnoy’s fixative, and incubated for 10 minutes at RT. The cells were spread onto a glass slide using a Pasteur pipette. The slides were air dried at RT. Purified DNA from Atm (RP23-456L9/9A5.3) was labeled with a red-dUTP (AF594, Molecular Probes) and purified DNA from a chromosome 9 control (RP23-284E19/9A5.2) was labeled with a green-dUTP (AF488, Molecular Probes) by nick translation. The probes were combined and hybridized to interphase and metaphase cells derived from the two samples using routine cytogenetic methods. Cells were stained with DAPI and total of one hundred interphase nuclei were scored for the number of red and green signals per cell.