Immunostaining of Stages 13 and 14 to Visualize the Meiotic Spindle

This protocol was designed to carry out immunostaining on meiosis I

prometaphase (stage 13) and metaphase (stage 14) oocytes to study the organization of spindles. The protocol is based on staining of the anterior half of mature eggs, obtained after cutting them through the anterior-posterior axis.

1. For each staining, dissect out with fine-tipped forceps 10-20 pairs of ovaries from well-fed females and place in 2 mL of absolute methanol kept in a small

Petri dish. Remove all the adhering tissues from the ovaries and gather the ovaries in another small Petri dish containing 1 mL of absolute methanol (see Notes 2-5 and 32).

2. Dissect out stage 13 and 14 oocytes from the ovaries by disrupting the ovarioles with tungsten needles.

3. By holding the dorsal appendages with fine-tipped forceps, cut the mature egg in half along its vertical axis with a scalpel or razor blade.

4. Remove the chorion and vitelline membrane from the anterior half of the stage 13 and 14 oocytes. Hold the dorsal appendages with fine-tipped forceps, and with a sharp tungsten needle, push out the anterior half of the egg from the chorion and vitelline membrane (see Note 33).

5. Immediately place the anterior half of the egg in a microcentrifuge tube containing 1 mL of absolute methanol. Collect approx 25-50 anterior egg parts.

6. Remove the methanol with a long-pulled glass Pasteur pipet, leaving 50 |L to cover the eggs (see Note 5).

7. Carry the sample through two consecutive quick washes with 1 mL of absolute methanol, removing the wash solution after each step. Use the long-pulled Pasteur pipet from step 6.

8. Add 950 |L of methanol to the microcentrifuge tube and rotate the samples for 2 h at a low speed on a rotating wheel (see Note 34).

9. Carry the sample through three consecutive quick washes with 1 mL of PBS, removing the washing solution after each step. Use a new long-pulled Pasteur pipet (see Note 35).

10. Carry the samples through three consecutive 5-min washes with 1 mL of PBT as described for step 9.

11. After the last wash, retain the ovaries in approx 50 ||L of PBT by removing most of the PBT with a long-pulled glass Pasteur pipet.

12. Follow Subheading 3.1., steps 15-27.

13. Transfer the egg parts and some mounting medium onto a microscopic slide with a short glass Pasteur pipet.

14. Cover the sample with a 24 x 24-mm2 cover slip, remove the excess mounting medium with 3MM paper, and seal the edges of the cover slip with nail polish (see Note 21).

15. Examine the microscopic specimen. Specimens can be stored in a dark place at 4°C (refrigerator, cold room) up to several months (see Note 22).

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