Wholemount antibody staining - adapted from Dan Weeks' protocol (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

Whole-mount Antibody Staining

(Adapted from Kolker, Tajchman and Weeks, Developmental Biology, 2000)

1. Fix embryos in Dent’s Fix for 2 hrs. (If needed, embryos can be stored in Dent’s overnight at 4oC.)

-Dent’s Fix = 80% MeOH + 20% DMSO

2. Bleach embryos in 95% Dent’s Fix + 5% H2O2 under a bright light on the rocker at RT 6 hrs. or overnight @ 4oC.

3. Wash embryos in Dent’s Fix 2X. (Embryos can be stored at -20oC in Dent’s overnight if needed.)

4. Rehydrate embryos:

5’ x 75% MeOH in PBS

5’ x 50% MeOH in PBS

5’ x 25% MeOH in PBS

5. Treat embryos in 1mg/mL hyaluronidase in 50mM acetate buffer for 45 min at RT.

-100μl 3M NaAc + 5.9mL H2O + 0.006g Bovine Testicular Hyaluronidase

6. Rinse 1X in PBS-TD

-PBS-TD = 49mL PBS + 0.5mL Tween 20 + 0.5mL DMSO

7. Block for 4 hrs. at RT on the rocker in PBS-TD + 0.1M glycine + 2% milk +5% serum

-Block = 12mL PBS-TD + 0.24 g milk + 0.6 mL lamb’s serum + 0.9g glycine

8. Incubate overnight at 4oC in the 1o antibody diluted in blocking solution.

9. Save the antibody if possible.

10. Wash embryos 6X in PBS-TD at RT for at least 1 hr. each wash.

11. Incubate embryos overnight at 4oC in the 2o antibody diluted in blocking solution. (Be sure to cover embryos in foil as the 2o antibodies are light-sensitive.)

12. Wash embryos 6X in PBS-TD at RT for at least 1 hr. each wash.

13. Fix embryos for 30 mins. in Dent’s Fix.

14. Store embryos in 100% EtOH at 20oC covered in foil.