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| With weak β-galactosidase expression, long incubation of glutaraldehyde-fixed embryos in staining solution results in an undesirably yellow embryo. It is possible to avoid this with this protocol. |
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1. Dechorionate embryos with 50% Bleach.
2. Rinse the embryos well with ddH2O and collect them on a Nitex mesh.
3. Blot the mesh on tissue paper to remove the excess water.
4. In a deep depression slide, fix embryos in 0.5 ml of Heptane that has been saturated with the fixative for 15 min at room temperature.
5. Cover the wells with a glass slide to prevent Heptane from evaporating. Embryos should turn light yellow. Whitish embryos are not well-fixed and will not stain well.
6. Transfer the embryos onto a glass slide using a Pasteur pipette.
7. Use a piece of filter paper to remove the excess Heptane. After all the Heptane has evaporated, take the embryos by gently touching with a doublesided-sticky tape and stick the tape on a glass slide, embryo side up (see Hint #2).
8. Cover the embryos with a drop of PBS.
9. Remove the PBS and add about 300 μl of staining solution without X-Gal (i.e. Fe/phosphate or Fe/CP). Incubate at room temperature for about 5 min.
10. Remove the liquid and replace it with the staining solution (see Hint #3). Incubate for 3 days at 30°C, refreshing the staining solution after 1.5 days.
11. Remove the staining solution (see Hint #4) and replace it with the PBS fixative.
12. Poke a hole in the vitelline membrane and let the PBS fixative penetrate the embryo for at least 10 min.
13. Devitellinize the embryos using a dissection needle (a tungsten needle is not necessary). You can use a sewing needle whose tip is sharpened with sand paper. Well-fixed embryos should be stiff and should come out from the membrane by gently scratching the vitelline membrane.
14. Transfer the embryos to a microcentrifuge tube.
15. Store the embryos in 90% Glycerol/PBS at 4°C (see Hint #5). They can be kept for months at this stage. Mount using two #1 coverslips as spacers.
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| X-Gal Solution |
| Store at -20°C
in DMSO (CAUTION! see Hint #1)
8% (w/v) X-Gal
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| CP Solution |
| 2.75 mM Citric Acid
200 mM Na2HPO4
pH 8.0
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| Fe/CP Solution |
| 5 mM Potassium Ferrocyanide (II)
5 mM Potassium Ferricyanide (III)
in CP solution
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| Fe/Phosphate Solution |
| 2.8 mM Sodium Phosphate Monobasic (NaH2PO4)
3 mM Potassium Ferrocyanide(II) (CAUTION! see Hint #1)
7.2 mM Na2HPO4
3 mM Potassium Ferricyanide(III) (CAUTION! see Hint #1)
1 mM MgCl2
150 mM NaCl
Store in the dark at room temperature
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| PBS Fixative |
| pH 7.2
add 4% (v/v) Formaldehyde
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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| Fixative |
| 1 ml of 0.1 M Sodium Cacodylate, pH 7.4
2 ml of Heptane
Shake the solution well and then let the phases separate. Use the Heptane layer (upper layer) for fixation. The lower layer can be reused by adding Heptane to 2 ml again.
1 ml of 18% (v/v) Formaldehyde (CAUTION! see Hint #1)
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| 90% Glycerol/PBS |
| pH 7.2
1.8 mM KH2PO4
4.3 mM Na2HPO4
2.7 mM KCl
add Glycerol to 90% (v/v)
137 mM NaCl
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| 50% (v/v) Bleach |
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| Staining Solution |
| Add one-thirtieth volume of X-Gal solution.
Warm up Fe/phosphate solution or Fe/CP solution to 37°C.
Use 200 μl to 300 μl per tube of embryos.
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Glycerol
DMSO
Heptane
X-Gal
Potassium Ferrocyanide (II)
Citric Acid
Potassium Ferricyanide (III)
Ethanol
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Cacodylate
Sodium Chloride
Formaldehyde
Sodium Phosphate, Monobasic
Potassium Phosphate, Monobasic
Bleach, Household
Magnesium Chloride
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Siliconized slides are easier to work with.
3. Staining with Fe/phosphate pH 7.2 and at higher temperatures (37°C) results in stronger staining, presumably because the conditions are closer to the optimum for the enzymatic activity. For ftz/lacZ fusion gene transformants, staining using Fe/CP overnight at room temperature is sufficient for visualizing stripes in the extended germband stage embryos and for CNS staining. To stain stripes at the blastoderm stage, use Fe/phosphate, pH 7.2 and stain overnight at 37°C. In this case, do not include older embryos in the same tube because they will stain too strongly and may transfer the bluereaction product to other embryos in contact. For the best resolution, try to use conditions that allow you to stain overnight, rather than stopping the reaction after a short incubation period. Staining at 37°C will increase the size of the reaction product crystals and will make identification of stained cells more difficult.
4. White precipitate will form in the staining solution during incubation. This does not affect the staining reaction, but it will stick to the embryos. Vortexing the embryos gently in Ethanol helps to remove it from the embryos.
5. Embryos can also be dehydrated and mounted in Epon for permanent preparations. GMM cannot be used because blue dye appears to dissolve (although slowly) in this medium.
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