Alcian green stain for cartilage Reference: Tuan, R. 1987. Mechanisms and regulation of calcium transport by the chick embryonic chorioallantoic membrane. J. Exp. Zool. [Suppl.] 1: 1-13. 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.   For example, click here.   Alcian green stain: 0.1 g Alcian green 70 ml 100% ethanol 1 ml conc. HCl 29 ml dH2O (100 ml)