This protocol gives excellent results on germarial stages. Fusome-specific antibodies have performed well, as has the BX69 antitubulin antibody to visualize spindle microtubules of the premeiotic divisions (see Fig. 4). Many of the ring canal proteins have also been successfully visualized using this protocol.
1. For each staining, dissect out with fine-tipped forceps 5-10 pairs of ovaries from females into 1 mL of EBR solution kept in a small Petri dish. Leave the very end of the abdomen attached to the ovaries to make them more visible during the immunostaining procedure, but try to remove as much fat body tissue as possible (see Notes 1-3).
2. Transfer the dissected ovaries into a microcentrifuge tube containing 1 mL of EBR solution (see Note 4).
3. Make the ovaries sink to the bottom by flicking the microcentrifuge tube a few times with your fingers.
4. Retain the ovaries in about 50 ||L of EBR solution by removing the rest with a long-pulled glass Pasteur pipet.
5. To carry out the fixation, add into the microcentrifuge tube 100 |L of buffer B fixative and 600 | L of heptane in the indicated order (see Note 5).
6. Shake the microcentrifuge tube gently by hand for 5 min (see Note 6).
7. Remove all the fixative with the long-pulled glass Pasteur pipet used in step 4.
8. Carry the ovaries through three consecutive washes with PBS by removing the washing solution after each step. Use a new long-pulled Pasteur pipet. Each wash should last at least 3 min. Place the microcentrifuge tube on a rotating wheel and rotate the sample at low speed.
9. Carry the ovaries through three consecutive 5-min washes with PBT.
10. Wash the ovaries in 1 mL of PBT for 1 or 2 h and leave the sample on the rotating wheel.
11. After this last wash, retain the ovaries in approx 50 |L of PBT.
12. Carry out the blocking by adding 900 |L of PBT and 50 |L of FCS (stock solution) into the microcentrifuge tube. Put the microcentrifuge tube on a rotating wheel and rotate at low speed for 30 min at room temperature (see Notes 7 and 8).
13. After blocking, remove the PBT-FCS solution with a new long-pulled glass Pasteur pipet and leave approx 50 |L of solution over the ovaries.
14. Repeat steps 12 and 13.
15. Dilute the primary antibodies with PBT in a total volume of 1 mL and add this solution to the microcentrifuge tube with ovaries.
16. Incubate the ovaries with the primary antibodies overnight at 4°C on the rotating wheel. From this step on, do not shake the sample. Pipet the solutions carefully into the microcentrifuge tube and wrap the tube in aluminum foil (see Notes 9-11).
17. Carry the ovaries through three consecutive 5-min washes with PBT, rotating the microcentrifuge tube at low speed. Use a new long-pulled Pasteur pipet to remove each wash solution.
18. After the last wash, remove all but approx 50 |L of PBT with the long-pulled glass Pasteur pipet used in step 17.
19. Dilute the secondary antibodies with PBS (1 : 200 v/v) in a total volume of 950 |L; then, add this solution to the microcentrifuge tube containing the ovaries (see Note 12).
20. Incubate the ovaries with the secondary antibodies at room temperature for 2 h on the rotating wheel. Protect the microcentrifuge tubes from light by wrapping in aluminum foil (see Notes 13 and 14).
21. Remove the secondary antibody solution with a new long-pulled Pasteur pipet.
22. Carry the ovaries through two consecutive 10-min washes with PBT on the rotating wheel at low speed. Remove the washing solution after each step using the long-pulled Pasteur pipet from step 21.
23. After the last wash, retain the ovaries in approx 50 | L of PBT, and then add 950 |L of PBS into the tube.
24. For DNA staining, add 1 |L of concentrated TOTO-3 to the microcentrifuge tube and incubate for 45 min at room temperature on the rotating wheel (see Notes 15-17).
25. Remove the diluted TOTO-3 solution with a long-pulled Pasteur pipet; then, quickly rinse the ovaries with 500 |L of PBS.
26. After this washing step, remove approx 450 |L of PBS with the long-pulled Pasteur pipet.
27. Add two to three drops of Vectashield mounting medium and incubate the samples overnight at 4°C in a dark place (see Note 18).
28. Transfer two to four ovaries and some mounting medium onto a microscopic slide with a short glass Pasteur pipet.
29. Dissect the ovaries into individual ovarioles and/or egg chambers with fine-pulled tungsten needles (see Notes 19 and 20).
30. 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).
31. Examine the microscopic specimen.
32. The microscopic specimen can be stored in a dark place at 4°C (refrigerator, cold room) for up to several months (see Note 22).
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