This procedure was used for the simultaneous hybridization of a human centromeric probe (FISH) and detection of the centrioles (immunostaining). Hybridization was first, followed by antibody detection for both the FISH probe and gamma-tubulin. A brief description of the procedure was also published in: Mantel et al. 1999 Blood 93(4):1390-1398.

Note:duration of antibody incubation (1:100 to 1:500 dilutions of a 1mg/ml stock) for both FISH and immunostaining is complete after 10 minutes at 37 C. Lomger incubations are not necessary. All antibodies were diluted in 4xSSC prior to placing them on the slide.

Slide preparation and fixation

Cells grown in culture, were placed on the slide using a cytospin. To permeabilize the cell membranes, slides were incubated in a 0.05% SDS in PBS solution for 2 1/2 minutes at room temperature. After incubation, slides were rinsed well in PBS, then passed for a few seconds each in 70% ethanol (to wash off salts) and 100 ethanol (dehydration) and then placed for a few seconds on a 75 C metal plate for quick drying.

Cells were fixed using the "chemical aging" approach, by keeping the slides for 30 seconds in ethanol at 95 C. After fixation, slides were air dried at room temperature.

Hybridization and detection

A digoxigenin-labeled DNA probe specific for the centromeric region of human chromosome 7 (Oncor, Inc) was mixed in Hybrisol VII (Oncor, Inc.) and was denatured 5 minutes at 75 C according to manufacturer’s protocol. The slides were denatured by placing them for 2 minutes in a 70% formamide/2x SSC solution at 75 C, followed by successive washes in 70%, 90% and 100% ethanol at room temperature.

Denatured centromeric probe was placed on slide, covered with a coverslip, sealed with rubber cement and incubated for 30 minutes to 2 hours at 37 C in a moist chamber.

After hybridization, slides were washed 3x5 minutes each in 50% formamide/2x SSC and 1xSSC solutions at 45 C.

For double staining, slides were incubated with a combination of antibodies against digoxigenin and gamma-tubulin as follows:

• in the first detection step, sheep-antidigoxigenin-FITC (Boehringer) and mouse-antitubulin (Sigma) were mixed together at 1:100 dilution each in a 4xSCC solution. 100 ul of this antibody solution was placed on each slide, covered with plastic coverslips and incubated 15 minutes at 37 C in a moist chamber. After incubation, the coverslips were removed and the slides were washed 3x5 minutes in 4xSSC/0.1% Tween 20 at 45 C.
• in the second step, a horse antimouse-TexasRed (Vector Laboratories) was used at 1:100 dilution in 4xSSC to detect the mouse antitubulin antibody. After 15 minutes incubation, slides were rinsed again 3x5 minutes in 4xSSC/0.1% Tween 20 at 45 C, and then briefly rinsed in distilled water to wash off the salt. Slides were air-dried at room temperature.
• Slides were covered with a DAPI antifade solution, covered with coverslips and examined under a fluorescent microscope (Leica Aristoplan) equipped with a cooled CDD camera (Photometrics) and image acquisition and processing software (Vysis, Inc) using a set of appropriate filters (Chroma Technologies).

An example of this procedure is depicted in the figure below (normal cells). A DIG-labeled probe for the human chromosome 7 centromere was hybridized for 2 hours. Posthybridization washes were followed by the antibody detection step, using an antidigoxigenin antibody labeled with FITC (green) and an primari antibody against gammatubulin. A secondary antibody labeled with TexasRed (red) was used to detect the centrosome signals.