Thioflavine

Diagnostic histologic methods were performed on standard blocks of tissue that were fixed in 4% buffered formaldehyde and then either dehydrated and embedded in paraffin or cryoprotected and cut on a freezing, sliding microtome. Paraffin sections from the olfactory bulb and tract, anterior medulla (two levels anterior to the obex), anterior and mid-pons, mid-amygdala with adjacent transentorhinal area, anterior cingulate gyrus (1–3 cm posterior to the coronal slice containing the genu of the corpus callosum), middle temporal gyrus (at the level of the lateral geniculate nucleus), middle frontal gyrus (4–5 cm posterior to the frontal pole), and inferior parietal lobule were stained immunohistochemically for α-synuclein using a polyclonal antibody raised against an α-synuclein peptide fragment phosphorylated at serine 129, after epitope exposure with proteinase K. The process leading to the choice of immunohistochemical method, as well as details of the method, have been described in a previous publication (7 (link)). The density of α-synuclein-immunoreactive Lewy bodies and neurites in each of the above-mentioned brain regions was scored, for more than 90% of slides, by a single observer (TGB), without knowledge of diagnosis, as none, sparse, moderate, frequent and very frequent, using the templates provided by the Dementia with Lewy Bodies Consortium (66 (link)). The remaining slides were scored by trainees under the instruction of the primary observer. For the substantia nigra (SN), LTS was estimated using the same scoring method but on thioflavine-S-stained thick (40 micron) sections due to the standard laboratory practice of sectioning the SN in this manner for unbiased morphometric analysis.

Diagnostic histologic methods were performed on standard blocks of tissue that were fixed in 3.75% neutral-buffered formaldehyde and then either dehydrated and embedded in paraffin or cryoprotected and cut on a freezing, sliding microtome. Each case was first staged according to the Unified Staging System for Lewy body disorders with a standard set of brain sections stained with an immunohistochemical method for phosphorylated α-synuclein as previously described [8 (link)]. The Unified Staging System is a modification of the scheme first devised by the Dementia with Lewy Bodies Consortium [8 (link), 58 (link), 59 (link)].
Paraffin-embedded sections from multiple body sites (Table 1) were stained in an identical fashion as the brain sections, using a polyclonal antiserum raised against an α-synuclein peptide fragment phosphorylated at serine 129, after epitope exposure with proteinase K [34 (link)]. The process leading to the choice of immunohistochemical method, as well as details of the method, has been described in a previous publication [10 (link)]. In each body region, the density of α-synuclein-immunoreactive perikaryal neuronal cytoplasmic inclusions as well as puncta and neurites was scored, at the site of highest density, by a single observer (TGB) without knowledge of diagnosis, as none, sparse, moderate, frequent and very frequent, using the templates provided by the Dementia with Lewy Bodies Consortium [58 (link)]. The total number of body sites examined varied between subjects as an initially broad sampling scheme was progressively reduced to those regions showing a greater likelihood to have positive staining. To evaluate the relative frequency of immunoreactivity in the different body regions, paraffin sections on a single stained slide from each body site listed in Table 1 were used. Following this analysis, selected regions of interest were further evaluated with up to five additional paraffin sections and/or 80 µm thick formalin-fixed, frozen sections.
Alzheimer’s disease histopathology was staged and graded on 40 µm thick sections stained with the Gallyas method for neurofibrillary tangles and the Campbell–Switzer and thioflavine-S methods for senile plaques [15 (link)]. Braak’s neurofibrillary tangle stages and CERAD neuritic plaque densities were assigned as described [16 (link), 62 (link)].

An HCV Renilla luciferase (HCV RLuc) reporter construct was used to measure the effect of each compound on cellular HCV RNA levels. The replicon was a generous gift from Seng-Lai Tan. In HCV RLuc, the HCV internal ribosome entry site (IRES) drives the translation of the neomycin and Renilla luciferase genes while the HCV nonstructural proteins (NS3 to NS5B) are translated from the Encephalomyocarditis virus IRES.^{29 (link)} The plasmid DNA was cleaved with Sca I, purified by phenol/chloroform extraction followed by ethanol precipitation, and used as template for RNA transcription using MEGAscript™ T7 RNA transcription kit (Ambion, Austin, TX). The RNA transcripts were treated with 2 U DNase I (Ambion) at 37 °C for 30 min, purified by acid phenol/chloroform extraction, followed by isopropanol precipitation, and suspended in diethylpyrocarbonate-treated water. RNA concentration was determined by spectrophotometry by measuring the OD₂₆₀. RNA integrity and size was checked on 1% agarose gel. Transcribed RNA was stored in aliquots at −80 °C until needed.
Huh-7.5 cells RNA were transfected with HCV RNA by electroporation. Briefly, subconfluent Huh7.5 cells were trypsinized, suspended in complete growth medium, and centrifuged at 1,000 × g for 5 min at 4 °C. The cell pellets were then washed twice with ice-cold phosphate-buffered saline (PBS) and suspended at 1.75 × 10⁷ cells/mL in ice-cold PBS. Replicon RNA (5 μg) was mixed with 0.4 mL of cell suspension and transferred to 2 mm gap width electroporation cuvette (Eppendorf AG, Germany) and pulsed with 5 times for 99 μsec at 820 V over 1.1 sec intervals using the ECM 830 electroporator instrument (BTX Havard Apparatus, Holliston, MA). After 5 min recovery period at room temperature, cells transferred to 10 ml complete growth medium, and seeded into 10 cm diameter cell culture dishes. Twenty-four hours after transfection, the medium was replaced with fresh complete DMEM supplemented with 1 mg/ml geneticin (Invitrogen) and the medium was replaced every three to four days with fresh medium containing 1 mg/mL geneticin. Geneticin-resistant colonies were selected for a period of two weeks and expanded in the presence of 250 μg/mL geneticin.
HCV RLuc replicon cells were seeded at a density of 10 × 10³ cells per well in 96-well plates and incubated for 4–5 h to allow the cells to attach to the plate. The compounds dissolved in dimethyl sulfoxide (DMSO) were added at a final concentration of 10 μM (DMSO solvent final concentration was 0.5%) and the cells were incubated for 72 h at 37 °C under 5% CO₂ atmosphere. The effects of compounds on HCV replication were then assessed by measuring the Renilla luciferase activity in compound-treated versus DMSO-treated cells. At the end of the incubation period, the medium was aspirated and the cells were washed with 1 × PBS. The Renilla luciferase reporter gene assay was performed using the Renilla luciferase assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, the cells were lysed by addition of 50 μL of 1× Renilla luciferase lysis buffer followed by two cycles of freeze/thaw. The luciferase activity content of the lysate was measured with a FLUOstar Omega microplate reader instrument (BMG Labtech, Germany) after injecting 50 μL of luciferase substrate and reading for 5 s.