CHAPTER 8 - HISTOLOGICAL METHODS

Chromosome Spreads

(Source: C. Walker)

To prepare 24 h Embryos:

1. Dechorionate 24 h embryos.

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

3. Incubate at 28.5C for 90 min 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 min, transfer the dish to ice for an additional 8 min.

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

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

To prepare 24 h 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 1 min.

4. Triturate the suspended cells two times in a 50 ul wiretrol microcapillary.

5. Drop droplets of the cell suspension onto a slide, prewarmed to 50C, 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 watchglass, 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 50C for about 10 min to completely dry.

8. Stain in Giemsa for 30 min:

Giemsa:
4 mls Giemsa Stock (Sigma Diagnostics)
4 mls 0.5 M Na Phosphate pH7
200 mls distilled water 9. Rinse twice with dH2O.

To prepare blastulae:

1. At 60-100 min after fertilization, remove the chorions with pronase:
  • Drain eggs; add 5 ml of 0.5 mg/ml pronase for 3.5 min
  • 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 min after fertilization, transfer the cells to 1.1% Na citrate at RT for 10 min.

    5. Fix in 3:1 methanol:acetic acid for 60 min 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 50C slide warmer.

    5. Stain with Giemsa for 30 min (see above).

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