Fixation of embryos:

Reagents:

• MEMFA

1 part 10x MEMFA salts

1 part 37% formaldehyde         

8 parts water

• Ethanol

Process:

1. Fix embryos in MEMFA for 1-2 hours at room temperature.
2. Embryos may be dehydrated into ethanol (after several washes) for long term storage at –20°C or used for cartilage staining.

Day 1:

Reagents:

• 100% Ethanol
• Alcian Blue Solution

20 mg Alcian Blue

15 ml Acetic Acid

35 ml 100% Ethanol

Note: 10 mg of Alcian Blue can be used for a lighter stain, but we have found that 20 mg of Alcian Blue produces darker, sharper images.

Process:

A. Dehydrate embryos into EtOH – 5 minutes each

1.   

2.   

3.   

4.   

5.    

B. Stain in Alcian Blue Solution for 3 nights 
Note: This period may be varied depending on the desired intensity of the stain, but we have found that 3 nights produces darker, sharper stains.

Day 5:

Reagents:

• 95% Ethanol
• 2% Potassium Hydroxide (KOH)
• Glycerol

Process:

A. Rinse in 95% Ethanol – 15 minutes each

1.  

2.   

3.  

B. Rehydrate in 2% KOH – 10 minutes each

1      75% EtOH, 25% KOH

2      50 % EtOH, 50% KOH

3      25% EtOH, 75% KOH

4     100% KOH

5     100% KOH

6     100% KOH

C. Wash through glycerol in 2% KOH – at least 1 hour each

1      20% glycerol in 2% KOH

2      40% glycerol in 2% KOH

3      60% glycerol in 2% KOH

D. Wash overnight in 80% glycerol in 2% KOH

1      80% glycerol in 2% KOH

E. Dissect out facial skeleton if desired.  This allows for much greater visualization of cartilage formation.

F. Storage of stained embryos and cartilages may be stored in 80% glycerol in 2% KOH.

Protocol adapted by Annie Chou and Tim Grammer