DIFFERENTIAL STAINING OF CARTILAGES AND

BONES IN THE FETUSES

Introduction
Staining refers to the addition of a dye which can offer contrast to the tissue
structures which can be then appreciated for anatomical investigations. The process of
differential staining involves the addition of two separate dyes under the same process
in order to generate the different contrast for different tissue types so that their relative
anatomy can be studied. This process is employed in the research related to fetal
ossification and development.
The purpose of studying the fetal skeletal development is to appreciate the affects
of various pharmacological agents that may render teratological changes, or to study the
nutritional impact of a specific feeding in the prenatal stage. Although these methods
have a very rare application as the process is intricate and difficult to yield results. It is
mostly applied to study the mice skeletal development but work on the chick embryo has
also been done respectively.
Since 1897 different protocols have been proposed, each of them has had some
defects. During this period Alizarin red for bones as it has a high calcium affinity,
toluidine blue for cartilages and then by alcian blue for cartilages has been employed.
Nowadays the alizarin red and alcian blue combination is applied mostly for the fetal
skeletal studies.
Method
The choice of the fetal sample is based upon the species and the developmental
stage which demonstrates the process of ossification. For example, 20 days postcoital in
the case of mice.
The embryos are collected and then eviscerated. They are fixed in the 90% or
Absolute alcohol. Cartilages are stained with 0.01% Alcian Blue stain prepared in
ethanol and acetic acid. Gradually the sample is rehydrated with descending degree of
alcohol. Clearing agent is used in the form of potassium hydroxide.
Bony parts are stained with 0.01% Alizarin red followed by rinsing of the
potassium hydroxide. Then treated with ascending series of glycerol in 1% potassium
hydroxide. In the end, stained embryo treated with pure glycerol with added crystal
tymol.
Observations
Bones and cartilages as skeletal parts become red and blue respectively. Due to the
above-mentioned clearing process the skeletal parts can be viewed clearly. Usually at the
time of staining the other left over soft tissues do not develop the stained color.
Conclusion
Osteogenesis study in laboratory animals requires differential staining which is
done by using two colors, alcian blue for cartilages and alizarin red for bones. It is
recommended to use colors in separate stages in order to give optimal stain.
It is better to solve Alcian blue which is used for cartilage staining in ethanol and
glacial acetic acid instead of water. Some kind of the alcian blue is insoluble in water and
sedimented, so the cartilages cannot gain any color.
The important point of clearing stage is that embryos and early postnatal samples
don’t require enzyme digestion and the best clearing process can be done by use of
potassium hydroxide. 1% KOH is the best clearing agent for these samples.
If the samples remain in fixative solution longer, the best result will be gained in
clearing stage. So, the fixation step can be lengthened in ethanol and have the more
transparent stained samples at last.
Alizarin red absorption by bones affected by the amount of Ca in bone structure.
Embryos bones in which calcification is started newly and have a little amount of
calcium, fixing in formalin solution such as formalin acid or neutral formalin result in
bone decalcification so it will reduce the stain affinity.
The evidence from this study suggests that it is better to use ethanol as fixation,
1% KOH as clearing agents in embryos and early postnatal staining.

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