Chromosomes Spreads

(Source: C. Walker from Zebrafish Book 5th Edition)

To prepare 24 hpf embryos

1. Dechorionate 24 hpf embryos.

2. Transfer into 10-2 M (4 mg/ml) freshly made colchicine.

3. Incubate at 28.5°C for 90 minutes in the dark.

4. At room temperature (~21°C), rinse the embryos and transfer embryos into 1.1% NaCitrate. Puncture the yolk and start a timer.

5. Dissect away the yolk.

6. At 8 minutes, transfer the dish to ice for an additional 8 minutes.

7. Transfer the embryos with as little citrate as possible into a 3:1 mixture of methanol:acetic acid.

8. Let stand 20 minutes and change the methanol acetic solution. Store in freezer overnight.

To prepare 24 hpf spreads

1. Pick up an embryo with forceps.

2. Blot it until partially dry.

3. Transfer the embryo into a watch glass containing 3 drops of 50% acetic acid and mince with forceps for 1 minute.

4. Triturate the suspended cells two times in a 50 μl Wiretrol microcapillary.

5. Drop droplets of the cell suspension onto a slide, pre-warmed to 50°C, and quickly pull the liquid back up into the Wiretrol. Drop ~6 droplets per slide.

6. Expel the remaining liquid in the Wiretrol onto the watch glass, mix and drop onto another slide, etc. for about 4 or 5 slides per embryo. This procedure will gradually dilute the cells yielding a range of cell densities on the slides.

7. Leave slides at 50°C for about 10 minutes to completely dry.

8. Stain in Giemsa for 30 minutes:

Giemsa:

4 ml Giemsa Stock (Sigma Diagnostics)
4 ml 0.5 M Na Phosphate pH 7
200 ml distilled water

9. Rinse twice with dH2O.

To prepare blastulae

1. At 60-100 minutes after fertilization, remove the chorions with pronase:

• Drain eggs; add 5 ml of 0.5 mg/ml pronase for 3.5 minutes
• Dilute eggs with 200 ml of 8x water. Rinse three times more with 200 ml washes of 8x water.

8x water = 12 ml stock salts per liter dH2O
Stock salts = 40 g Instant Ocean® per liter dH2O

2. Transfer the embryos into 3.5 cm Petri dishes at a density of 25 per dish in 8x water.

3. Break the yolks by lifting the embryos to the surface on a sharp probe. This will produce a ball of cells.

4. At 120 minutes after fertilization, transfer the cells to 1.1% Na Citrate at room temperature for 10 minutes.

5. Fix in 3:1 methanol:acetic acid for 60 minutes and then change to fresh 3:1.

6. Store in freezer.

To prepare blastulae spreads

1. Transfer the blastulae in 3:1 onto an ice cold slide.

2. Tease apart with fine needles or forceps.

3. Add small droplets of 50% acetic acid and flame the slide three times over a Bunsen burner.

4. Transfer the slide to a 50°C slide warmer.

5. Stain with Giemsa for 30 minutes (see page 8.26).