A simple procedure for rehybridization of nuclei analyzed previously by fish

Abstract

Fluorescence in situ hybridization (FISH) provides a direct way of detecting specific DNA sequences in metaphase chromosomes and interphase nuclei. This technique has rapidly found applications in clinical and cancer research. In most FISH studies, fresh or stored fixed cells are used for hybridization experiments, and the slide is used only once (Wang-Rong et al., 1995). We developed a simple procedure for FISH by which the same slide can be used for two consecutive FISH experiments with different detection systems and fluorochromes.Tumor specimens obtained after needle biopsies from patients with prostate cancer were processed according to standard procedures (Brown et al., 1994;Dierlamm et al., 1996). In brief, the samples in saline solution were mechanically disaggregated and treated with hypotonic sodium citrate solution (1%) for 20 min and methanol/acetic acid (3:1) overnight. The remaining cell clumps were disaggregated with 60% acetic acid and the suspension was dropped onto cold slides which were stored at -70 o C until used. FIRST HYBRIDIZATIONSlides were pretreated with 2 x SSC, pH 7.0-7.4, for 30 min at 37 o C, dehydrated in a graded ethanol series (70, 85 and 100%; 2 min each) at room temperature and air dried. An alternative pretreatment was also used as follows: the slides were washed in 70% acetic acid for 1 min at room temperature and three times in 1 x PBS for 4 min each at room temperature. Then, 100 µl DNAase-free RNAase A (Sigma; 100 µg/ml in 2 x SSC) was dropped onto a plastic coverslip, which was then placed over the pretreated slides and incubated for 45 min to 1 h at 37 o C in a moist chamber with 2 x SSC, pH 7.0. After three washes for 5 min each in 2 x SSC, pH 7.0, dehydration in a graded ethanol series (70, 85 and 100%; 2 min each) and air drying, the slides were incubated in pepsin solution (Sigma; 10 mg in 100 ml 0.01 M HCl) for 5-10 min at 37°C and washed in 1 x PBS (5 min). After incubation in 4% formaldehyde/1 x PBS for 10 min at 37°C, the slides were washed in 1 x PBS (5 min), dehydrated in a graded ethanol series for 2 min each and air dried.Cellular DNA was denatured by immersing the slides in 70% formamide/2 x SSC, pH 7.0-7.4, for 2-4 min at 72°C, followed by dehydration in a cold ethanol series (-20 o C). Five microliters of hybridization mixture (30 µl Hybrisol VI Oncor and 1.5 µl centromeric probe) was denatured for 5 min at 72-74°C. After brief cooling on ice, this mixture was added to each slide, which was then covered with an 18 x 18 mm coverslip and sealed with rubber cement. Hybridization was allowed for 16-24 h in a moist chamber at 37°C. Then the slides were washed in 50% formamide and 2 x SSC for 5 min each at 37°C. After incubation in phosphate Tween 20/PBT (30% BSA, 1% Tween 20/PBS) or phosphate buffer detergent/PBD (Oncor) for 2 min at room temperature, the biotin-labeled probe was detected with fluorescein-labeled avidin at final concentration of 1:1000 wash buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% v/v Tween 20, 5% w/v non-fat dry milk powder), and th...