Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon Pathcentre, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.
Historically, some fetuses for regulatory developmental toxicity studies have been stained with alizarin red S and cleared with glycerol to visualize the ossified portion of their skeletons. Interest in examining cartilage arose owing to its inclusion in some regulatory guidelines. Methods for double staining rat skeletons have been published previously. The method described here for staining mouse skeletons is fully automated and uses alizarin red S to stain bone and Alcian blue to stain cartilage. Pregnant mice (Crl:CD1) were euthanized on gestation day 18 to obtain fetal specimens. Day 0 post-partum mouse pups also were stained. Our method was developed using the Shandon Pathcentre , which is a fully enclosed automated staining system that allows staining to be carried out at 30° C with a final clearing at 35° C. Our method uses the same solutions as for fetal rat processing, but with reduced time periods for the smaller size of mice vs. rat specimens. Staining, maceration and clearing of the specimens requires approximately 2 days. The time required of laboratory personnel, however, is minimal, because all solutions are changed automatically and the specimens do not require examination or removal from the processor until processing is complete. After processing, the specimens are suitable for immediate assessment of bone and cartilage. A mouse developmental toxicity study using 20 animals/group and approximately 10 fetuses/animal could be processed in only three runs using one machine.
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