All brains were examined in the Division of Neuropathology of the Johns Hopkins University. After weighing and external examination, the right hemi-brain is cut in 1-cm coronal slabs and a standard set of tissue blocks is removed for overnight fixation in 4% paraformaldehyde or snap freezing. The remaining coronal slabs are frozen on prechilled aluminum plates and then maintained at −80°C. The left hemi-brain is fixed in 10% buffered formaldehyde for at least 2 weeks and then cut coronally. For diagnostic purposes, tissue blocks are dissected from middle frontal gyrus, superior and middle temporal gyri, inferior parietal cortex, occipital cortex, cingulate gyrus, hippocampus, entorhinal cortex, amygdala, thalamus, basal ganglia, midbrain, pons, medulla, and cerebellum (Table 1). For diagnostic purposes, tissue blocks are processed and embedded in paraffin, cut at 10 μm, and stained with hematoxylin and eosin. Selected sections are silver-stained with the Hirano method [9 (link)] and immunostained for α-synuclein (BD Transduction Laboratories, Palo Alto, CA; dilution, 1:500) and phosphorylated tau (anti-phosphorylated tau, paired helical filament 1 clone; a gift of Dr. P. Davies, Albert Einstein College of Medicine, Bronx, NY; dilution, 1:100). In selected brains, we prepare fixed tissue sections for stereology. Presently, we are obtaining a set of large tissue blocks containing the entire temporal lobe including hippocampus and entorhinal cortex, and a set from the brainstem and lower diencephalon containing the entire substantia nigra and locus coeruleus.
The severity of neuritic plaques is assigned a semi-quantitative and age-adjusted score (0, A, B, or C) according to CERAD [10 (link)], and the distribution of neurofibrillary tangles is assigned a stage score (0–VI) according to Braak [11 (link)] (Table 1). Although immunostains for tau (PHF1) and the amyloid peptide Aβ (6E10, Signet Laboratories) are performed, all Braak and CER-AD staging is based on silver stains. The assessment of Lewy body diseases, specifically idiopathic PD and Dementia with Lewy bodies (DLB), is conducted on both H&E and α-synuclein stained tissue sections. For diagnosis of PD we follow the criteria of the London Brain Bank [12 (link)] and for DLB the criteria of the DLB consortium [13 (link)].