This document outlines a skeletal examination procedure involving double staining for ossified bone and cartilaginous tissue, which is vital for prenatal developmental toxicity studies of chemicals and pesticides. It details the materials required and the step-by-step process for skinnng, eviscerating, and staining fetuses using alcian blue and alizarin red S. References supporting the methodology are also provided.

1. Skeletal Examination Procedure by Double Staining
for Ossified Bone and Cartilaginous Tissue

2. Introduction
• The assessment of developmental toxicology data is
a critical aspect of hazard evaluation for
pharmaceuticals and environmental chemicals
• Skeletal examination is an essential part of prenatal
developmental toxicity studies of chemicals as well
as pesticides

3. Requirement’s
1. Ethanol 96% (Fixative)
2. Alcian Blue stain (2 g Alcian blue dissolved in 16
L ethanol 96% and 4 L glacial acetic acid)
3. Alizarin Red S (1 g Alizarin Red S dissolved in
100 mL deionized water)
4. KOH solution (1, 1.5, or 2%)
5. Glycerol (industrial grade)
6. Deionized water
7. Stereomicroscope
8. Petri dishes

4. Skinning and Evisceration
fetuses are skinned to allow the Alcian Blue to
penetrate
skin is gently peeled off without cutting using a pair of
tweezers
After skinning the fetuses are eviscerated

5. Clearing and Staining
1. Fix in ethanol for about 4 weeks
2. Alcian blue stain for 48 h
3. Alcian blue stain is then replaced with ethanol for 72 h
4. Ethanol is decanted and replaced with 1% KOH
solution for 48 h
5. Fill of the staining jar with 1% Alizarin Red S + 1%
KOH solution for 48 h
6. Store in Glycerol solutions

6. Images

7. References
• Kimmel CA, Trammell C (1981) A rapid procedure for routine double staining of
cartilage and bone in fetal and adult animals. Stain Technol 56:271–273.
• Beltrame D, Giavini E (1990) Morphological abnormalities in experimental
teratology: need for a standardization of current terminology. Cong Anom 30(3):187–
195.
• Chahoud I, Buschmann J, Clark R et al (1999) Classification terms in developmental
toxicology: need for harmonisation. Reprod Toxicol 13:77–82.
• Paul C. Barrow (ed.), Teratogenicity Testing: Methods and Protocols, Methods in
Molecular Biology, vol. 947, DOI 10.1007/978-1-62703-131-8_17, © Springer
Science+Business Media, LLC 2013.

8. Thank you