FISH of Polytene Chromosomes

3.4.1. Preparation of the Slides for FISH

1. Wash the slides three times in 2X SSC, 5 min each wash (if the epitope-antibody interaction survives FISH, perform all the steps in the dark).

2. Dehydrate the chromosomes by passing the slides twice in 70% ethanol, 5 min each wash, and twice in 96% ethanol, 5 min each wash.

3. Allow the slides to air-dry and store at 4°C overnight (maintain the slides in a flat position to favor flattening of the chromosomes).

3.4.2. Preparation of the Hybridization Probe

1. Label 1 |ig of DNA by biotinylation using the BioNick (BRL) nick-translation kit, according to manufacturer's instructions.

2. After the nick-translation reaction, add to the sample 1/10 vol of 3 M sodium acetate, pH 5.3, and ethanol precipitate by adding 2.5 vol of cold 96% ethanol and incubating at -80°C for 1 h or at -20°C overnight. After centrifugation in a

4°C microfuge at maximum speed for 30 min, wash the biotinylated DNA pellet once with 70% ethanol, dry the pellet, and dissolve in 20 ||L of TE.

3. Mix the solution of biotinilated DNA with 200 |L of hybridization buffer. The mixture is stable at -20°C.

3.4.3. Hybridization

1. If the antibody staining survives FISH, perform all steps in the dark. Just before hybridization, incubate the slides in 2X SSC for 45 min at 70°C (do not put slides into hot SSC; instead, put them into SSC at room temperature and then put the slide rack into a 70°C water bath). Dehydrate by passing through 70% ethanol (twice for 5 min each) and 96% ethanol (twice for 5 min each). Air-dry the slides.

2. Denature the DNA by incubating the slide in 100 mM NaOH for 10 min. Wash the slides three times in 2X SSC (1 min, 1 min, and 5 min) and dehydrate by passing through 70% ethanol (twice for 5 min each) and 96% ethanol (twice for 5 min). Air dry slides.

3. Denature an aliquot (approx 10 |L per slide to be hybridized) of the hybridization mixture containing biotinylated probe DNA for 5 min at 80°C, then snap-cool on ice. Prewarm the hybridization mix again to 37°C. Load 6-10 |L of the mixture on the slide, cover by a cover slip, and seal with rubber cement in order to prevent liquid evaporation. Hybridize overnight in a dark humid chamber at 37°C.

3.4.4. Washing and Detection

1. After hybridization, remove the rubber cement and then remove the cover slips by immersion in one bath of 2X SSC.

2. Wash in 2X SSC three times at 42°C, 5 min each, and then 5 min at RT.

3. Add 40 |L of FITC-conjugated streptavidin (for green staining) or Texas Red-conjugated streptavidin (for red staining). Dilution ranges from 1 : 30 to 1 : 100 (to be tested by a serial dilution experiment) in the detection solution.

4. Incubate for 1 h at RT in a dark humid chamber. Perform all subsequent steps in the dark.

5. Wash in 2X SSC three times at RT, 5 min each, and once in PBS for 5 min. (Optional: The FISH signal from fluorescent-coupled streptavidin may be amplified using biotinylated anti-streptavidin antibodies; see Note 6.)

6. Stain the slides for 10 min in 40 |L of DAPI solution (0.5 |g/mL in PBS). Wash 5 min in PBS.

7. Mount with Mowiol and acquire FISH images at the fluorescence microscope.

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