Charles S., "Interleukin-1 polymorphisms associated with increased risk of gastric cancer" (2000). To evaluate dopaminergic cells of the dorsomedial cluster by tyrosine hydroxylase immunostaining, serial 4-mm sections were cut to include the entire brain. Immunopositive cells at the level of the giant interneuron commissure, posterior to the fan-shaped body, were counted in well oriented frontal sections at 1, 10, 30 and 60 days. At 1 day all control and experimental sections contained four or ®ve cells in the delineated region. At 30 and 60 days all controls showed four or ®ve cells. At 30 and 60 days all a-synucleinexpressing animals (a-synuclein, elav±GAL4 and a-synuclein, Ddc±GAL4 transheterozygotes) showed 0 or 1 tyrosine-hydroxylase-positive cell in the de®ned region. Tyrosinehydroxylase-positive cells outside the dorsomedial cluster were present, and served as internal controls for the immunostaining procedure. At least four, and usually between six and ten brains were examined for wild-type a-synuclein and each mutant a-synuclein. Controls included young and aged¯ies of the genotypes elav±GAL4/+ and Ddc±GAL4/+. We evaluated expression of a-synuclein and b-galactosidase on similar serial section preparations. Quanti®cation was simpli®ed in these experiments because no clear cellbody-associated a-synuclein or b-galactosidase immunoreactivity was observed in the aged a-synuclein transgenic¯ies at the times reported.For histological examination of retinas, heads were ®xed in glutaraldehyde and embedded in epon. Tangential retinal sections were prepared at a thickness of 1 mm and stained with toluidine blue (Fig 4).Standard electron microscopy was performed on brains from 25-day-old experimental (UAS±A30P a-synuclein/elav±GAL4) and control (elav±GAL4/+)¯ies. For immunoelectron microscopy, pre-embedding immunohistochemistry with an Hrp-congugated secondary antibody was performed on 60-day adult brains from experimental (UAS± A30P a-synuclein/elav±GAL4) and control (elav±GAL4/+)¯ies ®xed in 4% paraformaldehyde with 0.5% glutaraldehyde. Tissue was post-®xed in osmium and embedded in epon. Unstained ultrathin sections and ultrathin sections stained with uranyl acetate and lead citrate were examined. Climbing assayThe climbing assay was performed as described 19,20 . Forty¯ies were placed in a plastic vial, and gently tapped to the bottom of the vial. The number of¯ies at the top of the vial was counted after 18 s of climbing. Twenty trials were performed for each time point. The data shown represent results from a cohort of¯ies tested serially over 55 days. The experiment was repeated three times, with independently derived transgenic lines. Similar results were obtained from each experiment. The experiment was carried out under red light (Kodak Safelight Filter 1A). Control¯ies were of the genotype elav±GAL4/+. Experimental animals were of the following genotypes: (1) elav±GAL4/+; UAS±wild-type a-synuclein/+; (2) UAS±A30P a-synuclein/elav±GAL4; and (3) UAS±A53T a-synuclein/elav±GAL4.

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