Peichuan Zhang Answered Feb 5, 2014
Department of Biology, The Pennsylvania State University, USA
Hi Farzane,
We did these experiments a long while ago --- I just checked my thesis and found this note, "samples were further treated overnight with 1.0% trypsin prepared in buffer containing 30% saturated sodium borate", and "when limp and blue cartilage can be readily seen, samples were further treated overnight with Alizarin Red (Sigma, Cat# A-3882" prepared in 0.5% KOH".
So for mice of 30 days old, I'd recommend you to follow the procedures as described in the Bio-Protocol website here. Stain the cartilage with Alcian Blue first, and proceed to the next mineralized bone staining when you can see the cartilage clearly. Prepare a saturated sodium borate solution, and use 30% (volume) to prepare a 1.0% trypsin for digestion overnight. This should allow the Alizarin Red dye penetrate into the bones.
Best,
Peichuan