Following inoculation, mice were monitored for onset of prion disease signs by experienced observers. Observation of onset and progression of the subtle clinical disease in these experiments was very difficult, as the extremely long incubation periods overlapped with the natural lifespan of the mice. In many cases the slowly progressive neurologic signs were complicated with other age-related concurrent conditions. Mice were euthanized when they displayed consistent neurologic signs including tremors, head bobbing, weakness, ataxia, circling or kyphosis or when other non-neurologic conditions such as weight loss or neoplasia necessitated euthanasia. Following euthanasia brains were removed, and half of the brain was placed into formalin and half of the brain was frozen for biochemical analysis.