On the day preparations: (allow 2-3 hours)
Solutions & preparation of materials:
1. 1% formaldehyde solution is made freshly from paraformaldehyde (PFA). Note that PFA is a polymer of formaldehyde that must be hydrolyzed to be functional. This is done by heating the aqueous solution (60°C) and increasing the pH with NaOH.
a. Add 0.25 g of PFA to 22.5 ml of double-distilled water in a 50 ml conic tube. Add one drop of 1 M NaOH. Incubate in 60°C water bath for at least 20 minutes. Invert every 5 minutes or so to ensure all PFA is dissolved.
b. Allow solution to cool to room temperature before adjusting the pH to 9.2 using 50mM boric acid (usually takes about 0.5 ml).
c. Add 35 µl of Triton X-100 (0.15% final v/v). Make sure is completely dissolved before use. Triton X-100 is a nonionic detergent that bursts the nuclear envelope. This is done concomitantly with the formaldehyde fixation step.
d. Add 150 µl of a 500 mM solution of DTT to the fix (final concentration: 3 mM DTT)
2. Thaw out Tyrode’s solution aliquot and pre-warm to 37°C.
3. Pre-warm aliquot (approx. 1 ml) of 0.9% sodium citrate solution to 37°C.
4. Prepare a humid chamber with hot water before beginning protocol.
5. Etch and clean microscope slides. Use a diamond pen to draw a circle in the middle on the underside of the slide where you will drop the oocyte. Use ethanol or IMS to wipe the slides to remove grease using standard laboratory tissue wipes.
The following steps are carried out under the stereomicroscope on a heated stage.
6. Place the slides in a Coplin jar containing in 1% formaldehyde solution to coat the slide.
7. Using a 130 µm micropipette, transfer the MI or MII oocyte to a 30 µl drop of Tyrode’s solution on the lid of a culture dish. Gently pipette the oocyte up and down to move the oocyte in the solution. Watch for the loosening and eventual sloughing of the zona under a stereomicroscope.
8. Once the zona sloughs off, carefully transfer the eggs though 2-3 drops of 0.9% sodium citrate to rinse off the Tyrode’s solution.
9. Pick up and hold the oocyte in the micropipette in the smallest amount of liquid possible.
10. With your other hand remove slide from the formaldehyde, dab the end on a paper towel and wipe the back to remove excess solution, and place under the stereomicroscope.
11. While looking under the stereomicroscope, drop the oocyte onto the formaldehyde-soaked slide within the etched circle, transferring the least amount of wash solution possible. Watch for the oocyte to visibly burst when it hits the slide. The oocyte will look to flatten and ‘crack open’, followed by a visible dispersion of the cytoplasm. Once completely burst the oocyte will be nearly invisible on the slide.
12. Taking care to keep the slide absolutely flat, transfer it to the pre-warmed humid chamber (37 °C) for gradual drying for a minimum of two hours at room temperature.
13. After slow drying, crack the lid of the humid chamber and leave until slides are fully dry at room temperature (approx. 20 minutes).
14. Move slides to a Coplin jar containing a 1% Photoflo solution (500 µL in 50 mL of water) and wash for 2 minutes.
15. Remove slides and air dry. Slides may dry standing vertically against any surface to allow excess liquid to run to the end of the slide or lying flat on paper towels. Leave at room temperature on the bench until completely dry (approx. 10-20 min).
16. Store slides -20°C until ready for staining.