Post-fixation horseradish peroxidase tracing in rat fetus and pups

Horseradish peroxidase (HRP) has been shown to be applicable to fixed tissue in tracing projections in adult animals. This technique has several advantages in studying short projections in difficult attainable brain areas, especially with fetuses in utero. We have used this method to trace developing fibers (e.g., vestibulo-cerebellar connections) in rat fetuses and pups. It has been possible to make discrete and precise injections in any location. The quality of labeling by this method is distinctly related to the grade of fixation. Fixation grade determines how well HRP is anchored inside fibers and cell bodies. In optimally fixed preparations fibers and cell bodies are intensively stained with HRP up to over 3 mm from the center of an injection site. HRP is probably only taken up by damaged fibers so that the amount of fibers labeled is determined by the extent of the lesion caused by the injection pipette.