Combined FISH and Immunostaining

1. Proceed as described in Subheadings 3.1-3.5.

2. After completing Subheading 3.5., step 6, rinse the embryos twice with PBST.

3. Block the embryos in approx 1 mL of 10% FCS in PBST for 1 h at room temperature. Mix gently (e.g., on a rotating wheel).

4. Remove the blocking solution and incubate the embryos in 1 : 10 to 1 : 100 mouse monoclonal anti-Drosophila lamin antibodies in 10% FCS in PBST for 4-6 h at room temperature or overnight at 4°C (see Note 14).

5. Wash the embryos in five changes of PBST, 30 min each wash.

6. Incubate the embryos in 1 : 100 FITC-avidin D and in an appropriate concentration of goat or donkey anti-mouse rhodamine-labeled secondary antibodies in 10% FCS in PBST, for 2-3 h at room temperature or overnight at 4°C (see Note 13).

7. Wash the embryos in five changes of PBST, 20-30 min each wash.

8. Mount the embryos in Vectashield or similar mounting medium and seal the cover slip with clear nail polish.

4. Notes

1. Gemkow et al. (2) have developed a simple technique to "paint" chromosome 4 using a combination of fluorochromes. YOYO-1 (a green-fluorescent dye; Molecular Probes) stains all chromosomes, whereas TOTO-3 (Molecular Probes) selectively stains chromosome 4. An advantage of this method is that it does not require denaturation of chromosomal DNA. However, it does require a light source capable of exciting TOTO-3 (e.g., 633 nm spectral line of a He-Ne laser) and an appropriate filter set.

a. Dechorionate and fix embryos as described in Subheadings 3.2. and 3.3.

b. Incubate embryos in 100 |g/mL RNase A in PBST for 3-5 h at 37°C.

c. Remove the RNase solution and replace with PBST containing 0.1 |iM YOYO-1 and 0.2 |M TOTO-3 for 30 min (at room temperature).

d. Mount in Vectashield mounting medium.

2. I have suggested biotin-dATP assuming an AT-rich satellite DNA is used as a probe; biotin-16-dUTP could also be used. Although hapten-conjugated dNTPs are normally efficient substrates for polymerases, they do not entirely substitute for their natural counterparts (see ref. 26 and references therein). This is particularly evident at high ratios of modified dNTP : unmodified dNTP, which can cause DNA synthesis to abort (26). For this reason and because not all incorporated biotin moieties will be accessible to avidin anyway because of steric hindrance, the biotin-dATP in the labeling reaction should be diluted with unlabeled dATP in a ratio of 1 : 4 (see Subheading 3.1., step 2). For AT-rich DNA probes, the amount of biotin-dATP could be reduced even further. Consider that the G-C-rich dodeca satellite (CCCGTACTCGGT) probe produces a strong FISH signal when labeled even with biotin-dUTP (13).

3. Fluorophore-labeled egg white avidin and bacterial streptavidin are available from numerous sources (e.g., ExtrAvidin®-FITC conjugate, Sigma-Aldrich). These are highly purified and chemically modified avidins with low nonspecific binding compared to the native proteins.

4. Biotin-14-dATP will be added just prior to the labeling reaction (see Subheading 3.1., step 2). If labeling with biotin-16-dUTP or digoxigenin-11-dUTP, for example, then dTTP would be left out of the mix instead of dATP.

5. Removal of unincorporated label helps to increase the signal-to-noise ratio. Etha-nol precipitation can be done as follows:

a. Transfer the stopped reaction to a 1.5-mL microcentrifuge tube and add 1 |L of 20 mg/mL glycogen (molecular-biology grade; e.g., Sigma-Aldrich, G 1767) and TE to bring the volume to 100 |L. Glycogen is suitable as a carrier for nucleic acid molecules as small as eight nucleotides (27).

b. Add 0.5 vol (50 ||L) of 7.5 M ammonium acetate and mix.

c. Add 2.5 vol (400 | L) of absolute ethanol, vortex briefly, and hold at -70°C for 1 h to precipitate the labeled DNA.

d. Centrifuge at maximum speed (12,000^-16,000^) in a microcentrifuge at 4°C.

e. Aspirate the supernatant, rinse the pellet in 1 mL of 70-80% ice-cold ethanol, and allow the pellet to air-dry (but do not overdry). Resuspend the DNA in an appropriate volume of formamide to give approx 2 ng/|jL (for satellite DNAs). Store the labeled probe at -20°C if not being used right away. After denatur-ation, add the probe DNA to an equal vol of 8X SSC, 0.2% Tween-20 to yield probe in 4X SSC, 0.1% Tween-20, 50% formamide suitable for hybridization.

6. Nick translation (21) can be used as an alternative method to label DNA. In this case, it is necessary to optimize digestion with DNase I to produce suitably sized probe fragments. Simple protocols for doing this in the context of FISH probes are described in refs. 28 and 29.

7. For short oligos and small DNA fragments, 3'-end labeling (or tailing) using TdT is required. A protocol for this can be found in (30,31).

8. Bleach and heptane will damage the brush, so dedicate a single paintbrush for this purpose.

9. This amount of probe (approx 25 ng) is sufficient for highly repetitive DNAs. Larger amounts are required for unique DNAs. For example, Gemkow et al. (8) used 600-1000 ng of DNA from a P1 clone.

10. Temperatures typically from 70°C-91°C are used to denature chromosomal DNA. For example, Hiraoka et al. (1) and Sigrist et al. (3) used 70°C for 15 min in 4X SSC, 0.1% Tween-20, 50% formamide. Gemkow et al. (8) used 80°C for 15 min in 4X SSC, 100 mM sodium phosphate, pH 7.0, 0.1% Tween-20, 50% formamide. Dernburg recommends denaturation at 91 °C for 2 min in 3X SSC, 10% dextran sulfate, 50% formamide (30).

11. A 37°C hybrization temperature should work for most probes, including dodeca satellite, 359-bp satellite, and unique DNAs. Lower temperatures may work better for A-T rich sequences. Lohe et al. (10) listed hybridization temperatures for satellite DNAs that may serve as a guide; bear in mind that they used a different hybridization solution.

12. If the embryo DNA is to be stained with a fluorophore that binds RNA as well as DNA (e.g., propidium iodide), include RNase A (0.5-1 mg/mL) at this step.

13. FITC-avidin D (1 : 100 dilution of stock) and FITC-anti-avidin D (1 : 100) can be added together to amplify the signal (13,32).

14. Immunostaining after the harsh conditions of FISH works well in the case of the lamin Dm protein, which is abundant in interphase and forms a stable polymer concentrated at the nuclear periphery. Dernburg and Sedat (31) reported that a surprising number of other proteins can also be immunostained after FISH, including tubulin, nuclear pore proteins, topoisomerase II, and GAGA factor.

15. Gemkow et al. (8) reported having successfully adapted the tyramide reaction for enhancing FISH signals (33,34) to whole-mount Drosophila embryos. Their protocol assumes a DIG-labeled probe was used, but biotin-labeled probes also can be detected by using streptavidin-horseradish peroxidase (HRP) conjugate (Perkin-Elmer Life Sciences):

a. Prepare the embryos as described in Subheading 3.7., steps 1-3.

b. Incubate with a 1 : 500 dilution of HRP-coupled anti-DIG antibody (Roche) for 3-4 h at room temperature or overnight at 4°C.

c. Remove antibody solution and wash the embryos extensively in PBST for 2-3 h, changing the wash solution every 20 min.

d. Remove the final wash and add tyramide buffer: 100 mM sodium phosphate buffer, pH 7.2, 10 mM imidazole, 5% polyvinyl alcohol (PVA) 30,000-70,000 MW (Sigma-Aldrich).

e. Add 0.2 ^g/mL Cy3-labeled tyramide (FITC-, TRITC-, and Cy5-labeled tyramides are also available; Perkin-Elmer Life Sciences) and incubate the embryos for 15 min at room temperature.

f. Start the peroxidation reaction by adding 0.001% H2O2. Incubate for 45 min.

g. Stop the reaction by washing the embryos in several changes of PBST.


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