Owing mainly to differences in race and fetal age, the procedures for dual staining of cartilage and bone in rodents, which have been used, cause a little trouble in applying them to the developing skeleton. Here, we present a simple and rapid procedure, a modification of previous ones, for staining of fetal skeleton from its formation until birth in mice and chicle that are frequently used in teratology research.
The procedure and results are summarized as follows;
1. Complete skinning and evisceration contributed to the whole procedure which yields high quality results.
2. Skinned and eviscerated specimen was stained and fixed in a mixture of 0.15% alizarin red S and 0.14% Alcian blue in ethanol and glacial acetic acid for 10 to 12 hours for cartilaginous skeleton but 15 to 20 hours for ossified one in both animals.
3. In time for tissue maceration; the longer, the better but it might separate the skeleton from the joint. So, time for it was prolonged stepwise from´6ne hour to 2, 3, 4, 6 and 8 hours in 2% KOH solution for the fetus at 14th, 15th, 16th, 17th, 18th and 19th day of gestation respectively in mice; and in chick, 30 minutes, 1, 2 and 3 hours in 1% KOH solution for the fetus aged 6, 7, 8 and 9 day and 2, 3, 4, 5, 6, 8 and 10 hours in 20o solution for 10, 11, 12, 13, 14, 15 and over 16 day respectively.
4. Subsequently, specimens in both animals were placed in 1 : 1 distilled water and glycerin for 12 to 14 hours then in 1 : 3 solution for 24 hours for clearing and hardening prior to final storage in pure glycerin.
High quality fetel specimens can be prepared for examination by this procedure within 30 hours and preserved for a long time.
|