Immunostaining of Stages 213

This protocol can be used to visualize many of the spectrosome-fusome and ring canal components in both the germarium and vitellarium. Cytoskeletal elements like the actin network are well preserved, and some anti-tubulin antibodies can be seen to decorate spindle microtubules during the cystocyte divisions.

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 ovaries to make them more visible during the immunostaining procedure, but try to remove as much as possible from the fat body tissue (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 approx 50 |L of EBR solution, removing the rest with a long-pulled glass Pasteur pipet.

5. To carry out the fixation, add 100 |L of buffer B fixative and 600 |L of heptane to the microcentrifuge tube in the indicated order (see Note 5).

6. Shake the microcentrifuge tube gently by hand for 10 min (see Notes 26 and 27).

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, removing the washing solution after each step. Use a new long-pulled Pasteur pipet. Each washing step should last at least 3 min. Attach the microcentrifuge tube to a rotating wheel and rotate at low speed.

9. After the last wash, retain the ovaries in approx 50 ||L of PBS (see Note 28).

10. Carry out the blocking by adding 900 |L of PBS and 50 |L of FCS (stock solution) into the microcentrifuge tube. Put the tube on a rotating wheel and rotate at low speed for 30 min at room temperature (see Note 8).

11. After blocking, remove the PBS-FCS solution with a new long-pulled glass Pasteur pipet, and leave approx 50 |L of solution on the ovaries.

12. Repeat steps 10 and 11.

13. Dilute the primary antibodies with PBS in a total volume of 1 mL and add this solution to the microcentrifuge tube with ovaries.

14. Incubate the ovaries with the primary antibodies overnight at 4°C. Put the microcentrifuge tube on the rotating wheel. From this step on, do not shake the sample, pipet solutions carefully into the tube and protect the tube by wrapping it in aluminum foil (see Notes 9-11).

15. Carry the ovaries through two consecutive washes with 1 mL of PBS, rotating the microcentrifuge tubes on a rotating wheel at low speed and removing the washing solution after each step. Use a new long-pulled Pasteur pipet. Each washing step should be 5-10 min.

16. After the last wash, retain the ovaries in approx 50 ||L of PBS.

17. Dilute the secondary antibodies with PBS (1 : 200 v/v) in a total volume of 950 | L and add this solution to the microcentrifuge tube containing the ovaries (see Note 12).

18. Incubate the ovaries with the secondary antibodies at room temperature for 2 h, rotating the microcentrifuge tube on a rotating wheel. Protect the microcentrifuge tube from light by wrapping it in aluminum foil (see Notes 13 and 14).

19. Remove the secondary antibody solution with a new long-pulled Pasteur pipet.

20. Carry the ovaries through two consecutive washes with 1 mL of PBS, removing the washing solution after each step. Use the long-pulled Pasteur pipet from step 19. Each washing step should last for 5-10 min, with the microcentifuge tube rotated at low speed.

21. After the last wash, retain the ovaries in approx 50 |L of PBS and add 950 |L of PBS to the tube.

22. Follow Subheading 3.1., steps 24-32, and substitute the PBT with PBS. 3.4. Immunostaining of Stages 1-13

This protocol gives excellent results for stages 1-13. It is based on paraformaldehyde fixation and the combined action of DMSO and di-PBT to make the ovarian tissue highly permeable to antibodies. Use this protocol if antibody penetration problems are encountered with the methods described in Subheadings 3.1.-3.3. Microtubule networks can be nicely visualized in the vitellarium, but the spindle microtubules are not properly preserved in the germarium. The actin network and the structures of the follicle cells, nurse cells, and oocyte nuclei are well preserved by this fixation method.

1. For each staining, dissect out with fine-tipped forceps 5-10 pairs of ovaries from females into 1 mL of di-PBT solution kept in a small Petri dish. Leave the very end of the abdomen attached to 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 to a microcentrifuge tube with 1 mL of di-PBT kept in an ice bath (see Note 4).

3. Make the ovaries sink to the bottom by flicking the microcentrifuge tube a few times with your fingers.

4. Remove all the di-PBT from the microcentrifuge tube with a long-pulled glass Pasteur pipet (see Notes 29 and 30).

5. Carry the ovaries through three quick consecutive washes with 1 mL of PBS, removing all the washing solution after each step. Use a new long-pulled Pasteur pipet (see Note 29).

6. To carry out the fixation, add 350 |L of PBS, 150 ||L of 10% PP solution (freshly prepared), and 600 |L of heptane to the microcentrifuge tube in the indicated order (see Note 5).

7. Shake the microcentrifuge tube vigorously by hand for 1 min and then leave the tube in a rack for 30 s to allow the two phases of the fixative mixture to separate (see Note 31).

8. Leave 50 | L of the lower phase of the fixative in the microcentrifuge tube to cover the ovaries and remove the rest of the fixative mixture with a new long-pulled glass Pasteur pipet.

9. Immediately add 650 |L of PBS, 300 |L of 10% PP solution, and 50 |L of DMSO in the indicated order. Put the microcentrifuge tube on the rotating wheel and continue to rotate the samples for 20 min at a low speed (see Note 31).

10. Remove all the second fixative solution with the long-pulled glass Pasteur pipet used in step 8.

11. Carry the ovaries through two consecutive quick washes with absolute methanol, removing the washing solution after each step. Use a new long-pulled Pasteur pipet.

12. Add 950 |L of methanol to the microcentrifuge tube and continue to rotate the samples for 30 min at a low speed (see Note 31).

13. Carry the ovaries through three consecutive quick washes with PBS by removing the washing solution after each step. Use a new long-pulled Pasteur pipet.

14. Carry the ovaries through three consecutive washes with di-PBT by removing the washing solution after each wash. Each washing period should last 5 min (see Note 30).

15. After the last wash, retain the ovaries in approx 50 ||L of di-PBT.

16. Carry out the blocking by adding 900 |L of di-PBT and 50 |L of FCS (stock solution) to the microcentrifuge tube. Rotate the tube at low speed for 1-2 h at room temperature (see Notes 7 and 8).

17. After blocking, remove the di-PBT-FCS solution with a new long-pulled glass Pasteur pipet, and leave approx 50 |L of solution on the ovaries.

18. Dilute the primary antibodies with di-PBT in a total volume of 950 |L and add this solution to the microcentrifuge tube containing the ovaries.

19. Incubate the ovaries with the primary antibodies overnight at 4°C. Put the microcentrifuge tube with ovaries on the rotating wheel. From this step on, do not shake the sample, pipet solutions carefully and protect the tube by wrapping it in aluminum foil (see Notes 9-11).

20. Carry the ovaries through three consecutive washes with 1 mL of di-PBT, rotating the tubes at low speed on a rotating wheel, and removing the washing solution after each step. Use a new long-pulled Pasteur pipet. Each washing step should last for 5 min (see Note 30).

21. After the last wash, retain the ovaries in approx 50 ||L of di-PBT.

22. Dilute the secondary antibodies with di-PBT (1 : 200 v/v) in a total volume of 950 | L and add this solution to the microcentrifuge tube containing the ovaries (see Notes 12, 13, and 30).

23. Incubate the ovaries with the secondary antibodies at room temperature for 2 h on a rotating wheel (see Note 14). Protect the tube from light by wrapping it in aluminum foil.

24. Remove the secondary antibody solution with a new long-pulled Pasteur pipet.

25. Carry the ovaries through two consecutive washes with 1 mL of di-PBT, rotating the microcentrifuge tube on a rotating wheel, and removing the washing solution after each step. Use the long-pulled Pasteur pipet from step 24. Each washing step should last for 10 min (see Note 30).

26. After the last wash, retain the ovaries in approx 50 ||L of di-PBT and add 950 |L of di-PBT to the tube (see Note 30).

27. For DNA staining, add 1 |L of concentrated TOTO-3 and incubate the sample for 45 min at room temperature on a rotating wheel (see Notes 15-17).

28. Remove the diluted TOTO-3 solution with a long-pulled Pasteur pipet and then quickly rinse the ovaries with 500 |L PBS.

29. Follow the instructions in Subheading 3.1. from step 26.

0 0

Post a comment