Immunofluorescence- Whole Mount

 

Objective:

Immunohistochemistry allows visualization of antigens (usually proteins) within an embryo. Typically, a primary antibody binds specifically to an antigen (e.g. Luciferase); then a secondary antibody conjugated to a flourochrome or enzyme is bound to the heavy chain constant region of the primary antibody with specificity to the species of origin of the primary (e.g. anti-rabbit conjugated to Rhodamine).

 

Procedures:

Start =>Staged, fixed, dechorinated embryos in PBST in siliconized 1.6mL microfuge tubes.
1. Remove PBST with pasteur pipette being careful to leave some residual fluid and embryos behind and undamaged. The same should be done prior to each wash below, unless otherwise stated.
2. Wash embryos 1 X 5'' (minutes) in PBST while rocking on nutator or similar low-impact shaker. ** Keep microfuge tubes almost full to lower stress to fixed embryos.
3. Wash embryos 1 X 5'' in H2O on nutator.
4. Permeabilize embryos by incubation in acetone at -20 degrees for 7''. ** Use acetone that is pre-chilled to -20 degrees.
5. Wash embryos 1 X 5'' in H2O. NO SHAKING, embryos will be fragile.
6. Wash embryos 1 X 5'' in PBST while rocking on nutator.
7. Incubate in immunoblock sol''n for 1.5+ Hrs while rocking on nutator at room-temp.
8. Dilute primary antibody in immunoblock (50 uL per sample, where each sample is ~20 or less embryos) ** Dilute according to best results of previous dilution series or use manufacturer''s suggested working dilution to approximate a dilution series to test for best dilution.
9. Withdraw all block sol''n from the embryos using a pulled pasteur pipette for the last ~100 uL. Be careful not do damage embryos.
10. Add 50uL of diluted antibody. Incubate at 4 degrees O/N. No rocking, etc.
11. Wash embryos 7 X 15+'' followed by 1 X 2 Hr in immunowash sol''n while rocking on nutator at room temp.
12. Incubate embryos 1 X 1.5+ Hrs in immunoblock while rocking on nutator at room temp.
13. Dilute secondary embryo in block sol''n (50 uL per sample, where each sample is ~20 or less embryos).
14. Withdraw all block sol''n from the embryos using a pulled pasteur pipette for the last ~100 uL. Be careful not do damage embryos.
15. Add 50uL of diluted antibody. Incubate at 4 degrees O/N. No rocking, etc.
16. Wash embryos 8 X 15+'' in immunowash while rocking on nutator at room temp.
17. Wash embryos 2 X 5'' in PBST. Embryos may be stored at this point for a few days until viewing on microscope.
18. Dehydrate embryos in methanol 2 X 10''. **Incomplete dehydration of the embryos will result in the unpleasent disintagration of the embryos when cleared.
19. Clear embryos by removing most of the methanol and adding 0.5mL of benzyl benzoate:benzyl alcohol :: 2:1. Mix gently, when embryos settle to the bottom suck up with a p1000 with a clipped tip (with large opening).
20. Load on depression slide and cover with 22mm X 22mm cover slip.
21. View with appropriate magnification and filter sets.

Reagents/Solutions

Immunoblock Solution

• 5 Goat serum in Immunowash solution.

  Immunowash

• 1 BSA; 1 DMSO in PBST.