Antigens Detection
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DETECTION OF ANTIGENS IN EARLY EMBRYOS (WHOLE MOUNT) USING
BIOTINYLATED SECONDARY ANTIBODIES
REAGENTS:
1. Blocking solution (Final concentration: 150 mM Maleic acid, 100 mM NaCl, 2%
Blocking reagent {Roche}, pH 7.5, 0.1% Tween-20)
2. ABC Vectastain Kit
3. DAB
4. 0.3 % H2O2 (made from fresh 30% stock with dH2O
5. PBST (Final concentration: 0.15M NaCl, 0.01M Sodium Phosphate buffer, pH
7.4, with 0.05% Tween-20. Autoclave at 121˚C for 15 minutes)
PROCEDURE
Fix Embryos using 4% PFA in PBS O/N @ 4˚C.
Wash 3X minutes with PBST.
Dechorionate the embryos if necessary (with watch maker forceps).
Permeabilize embryos by immersing in solutions with increasing amounts of
methanol. Wash 1X 5 minutes with: 30%  50%  70%  90% 2X in 100%
Methanol. Embryos can be stored in -20˚C til use.
5. Rehydrate by washing in 70%  50%  30  (Methanol in PBST), 5 minutes
each and a last wash 2X 3 minutes with PBST/PBT.
6. Block nonspecific sites by incubating in Blocking solution for 60 minutes at RT
or 4˚C O/N.
7. Incubate in 1˚ Antibody diluted, in blocking solution O/N at 4˚C.
8. Wash 4X 25 minutes with blocking solution.
9. Incubate with biotinylated antibody for 4 hours at RT or 4˚C O/N.
10. Wash 4X 25 minutes each with blocking solution.
11. During last wash prepare AB reagent by gently mixing 5 ul A solution (in 1 ml of
blocking solution) with 5 ul B, mix gently and let sit for 30 minutes without
shaking.
12. incubate embryos for 45 minutes in AB complex at RT (no longer than 1 hour).
13. Wash 3X 25 minutes with blocking solution and 1X 25 minutes with PBST.
14. Add 0.1 ml of DAB (concentration 20 mg/ml) to 1.9 ml PBST and incubate
embryos for 15 minutes in this solution.
15. Add 2 ul of 0.3% H2O2 to 1 ml DAB-PBST solution. Mix well and then stain the
embryos while monitoring the color formation. If the staining reaction is slow,
increase the amount of 0.3% H2O2 by factor of 5.
16. Stop the reaction by washing with PBST several times.
17. The specimen can be stored in 4% PFA, or in methanol (30%  70%  90% 
100% methanol).
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MOUNTING??
1. Methyl Cellulose mounting
2. Araldite mounting
01/02/07