CHAPTER 8 - HISTOLOGICAL METHODS
(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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