About a week ago, someone on this or the zebrafish list asked
about cartilage staining. I didn't save the message, but here is
a procedure that has worked well for us in the axolotl.
The staining procedure was adapted by K. Johnson (personal
communication) from the method of S.V. Bryant and L. Iten (1974).
After fixing, the embryos were rinsed twice with fresh 2% NH4OH
solution, and left at 4o C overnight. The embryos were then
dehydrated for 2 hours in 50% ethanol followed by another 2 hours
in 1% acid ethanol. The embryos were then placed in 1% Victoria
Blue B stain in 1% acid ethanol for 2 hours. Embryos were
destained with four rinses in 70% ethanol and left in 70% ethanol
overnight. Embryonic pigments were then bleached with two 24 hour
soakings in 10% hydrogen peroxide solution. After bleaching, the
embryos were dehydrated in two 30 min. rinses in 95% ethanol
followed by two more rinses in 100% ethanol. They were then
cleared and kept in methyl salicylate.
Bryant, S.V., and Iten, L. (1974). The regulative ability of the
limb regeneration blastema of Notophthalmus viridescens:
Experiments in situ. Roux's Arch. Dev. Biol., 174: 90-101.
John Armstrong e-mail: jbarm at acadvm1.uottawa.ca
Department of Biology
University of Ottawa
Ottawa, Ontario K1N 6N5