wholemount antibody staining - adapted from Dan Weeks' protocol (Conlon lab)
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Protocol submitted by VGP from Conlon lab protocols 
link to protocol document 
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.