CGH Protocols （四）

Images were acquired through a Zeiss Axiophot fluorescence microscope using a Plan NEOFLUAR oil objective x63, N.A. 1.25 (Zeiss, Oberkochen, Germany) equipped with filter sets appropriate for DAPI (Zeiss filter set 02, excitation: G365, beamsplitter: FT 395, emission: LP 420), FITC (Zeiss filter set 10, excitation: BP 450-490, beamsplitter: FT 510, emission: BP 515-565) and TRITC (Chroma filter set HQ Cy3+excitation filter from Zeiss filter set 15, excitation: BP 546/12, beamsplitter: FT 565, emission: BP 570-650) with a cooled CCD camera (Photometrics, Tucson, Arizona, U.S.A.) connected to a Macintosh Quadra 950 (Apple, Cupertino, California, U.S.A.). The resolution of this apparatus configuration is 0.108 m/pixel. The maximum image size is 1320x1035x12 bit.

The 100 W mercury lamp and the diaphragms of the microscope were precisely adjusted to get a homogeneous illumination of the optical field.

For each metaphase spread three monochrome images were digitized, one image for each fluorochrome. An image size of 512x512 or 768x768 pixels was chosen, according to catch the whole metaphase with one image. The images were inverted in order to make it possible to use a standard segmentation process and then transferred as 8 bit TIFF-files to a server PC via a local area network.

Image processing was carried out with the image analysis software AMBA on the basis of the standard karyotyping software KARYOTYP that was modified and extended for the CGH purpose. The software was installed on a 90 MHz Pentium PC, running Microsoft Windows (Microsoft Corp., Redmond, Washington, U.S.A.).

The program comprises the following steps:1) image segmentation using the DAPI image; 2) correction of optical shift; 3) computation of the fluorescence ratio images between FITC and TRITC images; 4) karyotyping; 5) determination of chromosome axis; 6) stretching of chromosomes and calculation of profiles; 7) evaluation.

附：

Normal chromosome metaphase spreads were prepared by standard procedures with special care to minimize cytoplasm debris. In general, no protein digestion was applied.

Prior to the hybridization, chromosome denaturation was performed for 60 sec at 77°C in 70% Formamid/2xSSC. Test and normal DNA, 5 µg each, were labeled by a standard nick translation reaction with Biotin and Digoxigenin, respectively. The DNase concentration was adjusted that the length of the labeled fragment varied between 200 and 1000 bp after 2 h incubation. One µg of each labeled genomic DNA, 20 µg of human Cot-1 DNA (Gibco BRL Life Technologies, Gaithersburg, MD, U.S.A) and 10 µg of herring sperm DNA were ethanol-precipitated.

The pellet was resuspended in 15 µl hybridization solution containing 33% Formamid/13.3% Dextransulfat/ 3xSSC, denatured at 77°C for 5 minutes and prehybridized at 37°C for 2 hours.

Finally, 12 µl of the hybridization mixture was applied to the slide with the denatured and dehydrated metaphase spreads. The hybridization was performed under a coverslip (18x18 mm) that was sealed with rubber cement for 3 days at 37°C. Detection of the differentially labeled genomic DNA was performed with avidin-FITC (Vector Laboratories, Burlingame, CA, U.S.A.) and anti-Digoxigenin-rhodamin (Böhringer Mannheim, Mannheim, Germany) followed by the DAPI staining for chromosome visualization. No enhancement reactions to increase the fluorescence signals of the genomic DNA probes were applied.