Tissue Harvest for Aspartoacylase Assays

Tissues from various regions of the rat brain, at key ages during postnatal development and maturation, were analyzed for aspartoacylase activity. The regions examined were: cerebral cortex, corpus callosum, cerebellum, hippocampus, brain stem, olfactory bulb and optic nerve at ages from postnatal day 1 (P1) to adult. Adult samples were taken from rats aged over 6 months. Various regions of the brain were removed sequentially and placed into cryovials. The optic nerves were sectioned just behind the eye and the nerve tracts dissected anterior to the optic chiasm. Olfactory bulbs were dissected out. The brain stem and cerebellum were also removed, then the cerebrum was resected from the skull and placed in a Petri dish containing ice-cold nutrient medium (Leibovitz L-15; Life Technologies, Paisley, UK). The dissociation of the meninges from the cortex, the dissection of the hippocampus and the separation of the corpus callosum was performed using a dissecting microscope. Initially, the corpus callosum was dissected from the rest of the structure was to leave the cortical sample. In this way, cross contamination between corpus callosum and cortex was minimized. Cortex, hippocampus and corpus callosum were washed by immersion in ice-cold phosphate-buffered saline in order to remove all possible contaminants from the dissection medium. Samples were placed in cryovials and immediately frozen in liquid nitrogen after dissection, and stored at -85oC until assayed. In all studies, at least six separate litters were used to provide brain material.

0 0

Post a comment