Alcian green stain for cartilage

This is an easy method for stianing cartilage that can be used with isolated or CAM-grafted limbs to study limb development or with whole embryos (8 to 12 days of incubation, ).

1. Fix limbs or embryos in 5% TCA for 1 hour at room temperature.
(Eviscerate embryos before fixation).

2. Wash 3X with PBS.

3. Immerse in Alcian green stain for 4 hours with gentle agitation.

4. Wash 3X with 70% ethanol, several hours to overnight.

5. Wash with 85%ethanol, 95% ethanol, 100% ethanol, several hours to overnight each.

6. Clear by washing 1-3X with methyl salicylate.

Alcian green stain:

0.1 g Alcian green

70 ml 100% ethanol

1 ml conc. HCl

29 ml dH2O

(100 ml)