Cartilage Staining

This protocol is adapted from one kindly provided to us by Dr. Carol Labonne.

Purpose: To use a basic water-based dye to stain for acid mucosubstances and acetic mucins located on the cartilage of fixed embryos, permitting the examination of cartilage formation.

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.

Cartilage

Protocol adapted by Annie Chou and Tim Grammer