Fixation of embryos:

Reagents:

MEMFA

10% 10X MEMFA salt

10% formaldehyde

80% water

Methanol

1) Fix embryos in MEMFA for 1-2 hr

2) Dehydrate in several MeOH washes

3) Embryos can now be stored long term

at -20°C

BEGIN Antibody Stain

DAY 1:

Reagents:

Methanol

PBT

1X PBS

2 mg/mL BSA

0.1% Triton X-100

1º Antibody

Rehydration:

5 minutes each wash

1) o MeOH

2) o 75% MeOH 25% H₂O

3) o 50% MeOH 50% H₂O

4) o 25% MeOH 75% H₂O

5) o 100% PBT

Blocking: (optional for 12/101)

1) o 5 min in PBT

2) o 5 min in PBT

3) o 1 hr in PBT

Primary Antibody:

o 1º Antibody 4 hr RT or 4°C O/N

DAY 2:

Reagents:

PBT

2° Antibody

PBT Washes:

1) o 10 seconds

2) o 20 min

3) o 20 min

4) o 20 min

5) o 20 min

6) o 20 min

Secondary Antibody:

1) o 2° Antibody 4 hr RT or 4°C O/N

DAY 3:

Reagents:

• PBT
• DAB

PBT+0.5 mg/mL DAB

• 1000X H₂O₂ solution
  1:1 dilution of 30% H₂O₂ in PBT
• Bleach

0.5X SSC

5% formamide

4% 30% H₂O₂ stock solution

PBT Washes:

1) o 10 seconds

2) o 20 min

3) o 20 min

4) o 20 min

5) o 20 min

6) o 20 min

DAB Detection:

1) o 10 min in DAB at 4°C

2) o Add 1X H₂O₂ solution to initiate reaction

3) Monitor reaction.

Usually takes 5-10 minutes. Reaction may be more optimal at 4°C rather than RT.

END Antibody Staining

1) o 5 min in PBT

2) o 5 min in PBT

3) o For a second stain, start over at 2°

Antibody after two 30 min PBT washes

Bleach:

1) o 2 hr in Bleach under fluorescent light

and then O/N at RT under ambient light