Immunofluorescent staining of mouse embryonic tissues

Embryo preparation

1. Dissect mouse embryos in ice-cold phosphate-buffered saline (PBS).

2. Fix embryos in 4% paraformaldehyde at 4°C overnight.

3. Wash embryos in PBS twice for 30 minutes each.

4. Wash embryos in saline for 15 minutes.

5. Wash embryos in 50% ethanol (in saline) for 15 minutes.

6. Wash embryos in 70% ethanol (in water) twice, 15 minutes each (embryos can be stored in 70% ethanol at 4°C).

7. Process the embryos into paraffin through a gradient ethanol-xylene-paraffin program in a tissue processor.

8. Embed embryos in paraffin.

9. Use a microtome and cut sections of the desired embryo regions at 5-7 µm thickness.

10. Carefully collect sections on Superfrost™ Plus microscope slides (Thermo Fisher).

11. Dry sections on slide warmer (42°C) overnight.

Deparaffinization and Rehydration

12. Immerse the slides in Xylene for 5 minutes, repeat once in second Xylene container.

13. Immerse the slides in 100% ethanol for 3 minutes, repeat once in second ethanol container.

14. Immerse the slides in 95% ethanol for 1 minute.

15. Immerse the slides in 80%, 70%, 50% ethanol successively for 1 minute each.

16. Rinse slides in H₂O for 1 minutes.

Antigen Retrieval (Microwave Method)

17. Soak the slides in citrate buffer for 5 minutes.

18. Microwave at highest power level until the liquid boils.

19. Switch Microwave to lower heat level for 15 minutes.

20. Let slides cool down in citrate buffer to room temperature (takes one hour or longer).

Blocking

21. Wash slides in PBS three times for 5 minutes each.

22. Remove slides from PBS and incubate tissue sections in blocking buffer for one hour at room temperature in a humid chamber.

Primary Antibody

23. Dilute the primary antibody to the recommended concentration in blocking buffer.

24. Add primary antibody solution onto tissues sections, cover with parafilm strip and incubate slides at 4°C overnight in a humid chamber.

25. Wash slides three times at 5 minutes each in PBS on a slow shaker.

Secondary Antibody and Detection

26. Dilute the appropriate fluorophore-conjugated secondary antibody in blocking buffer.

27. Add secondary antibody solution onto the tissue sections and cover with parafilm strip. Incubate slides for 1 hour at room temperature in a dark chamber.

28. Wash slides in PBS three times at 5 minutes each on a slow shaker.

29. Add antifade Mountant (e.g., ProLong^TM Diamond Antifade Mountant from ThermoFisher) over the sections on the slide.

30. Apply coverslip to slide and store slides at 4°C until ready to view.

Solutions:

4% paraformaldehyde in PBS

1x PBS

Saline

Ethanol (50%, 70%, 80%, 95%, 100%)

Xylene

Paraffin

Antigen retrieval buffer: 10mM citrate, pH 6.0

Block buffer: 2% donkey serum, 5% BSA, 0.1% tween-20, 0.2% triton x-100 in PBS

ProLong™ Diamond Antifade Mountant (Invitrogen, P36970)

Tested primary antibodies:

Goat anti-Foxf1 (R&D Systems, AF4798, 1:50)

Sheep anti-Foxf2 (R&D Systems, AF6988, 1:50)