Fig. 4: In vitro autoradiography of ³H-BU99008 in brain tissue from people with AD and controls.

A Greater ³H-BU99008 binding around Aβ plaques in AD brain tissue sections. Representative autoradiographs showing ³H-BU99008 total (left panel) and non-specific binding (centre panel; determined with 10 µM BU224) in 12 µm frontal cortex sections of the human brain (AD and age-matched controls). Standards (ARC 123B) represent a linear range of radioactivity. Immunohistochemistry for total Aβ (1 µg/mL pan-anti-Aβ, MOAB-2, clone 6C3, right panel) shows spatial distribution of plaques. White arrows point to high-intensity binding of ³H-BU99008 in the vicinity of plaques (black arrows), as magnified inserts show. B ³H-BU99008 binding is increased in AD brain sections relative to control brain. Comparative quantitative analysis of specific binding of ³H-BU99008 in grey and white matter (when identifiable) or total section of 12 µm frontal cortex sections (AD with Braak stages 5–6 and controls). Binding of ³H-BU99008 in the total sections from AD cases is significantly higher (unpaired t-test) than that in controls. Data is mean (±SD) from triplicates; AD: n = 6, Control: n = 5. C Non-specific binding is not different between AD and control brain sections. Quantitative analysis of non-specific binding, showing no difference between brain sections from AD and controls. Increased specific binding in AD (A/B) sections relative to controls therefore is not caused by differences in non-specific binding. D ³H-BU99008 binding colocalised with GFAP staining. Autoradiograph showing ³H-BU99008 total binding (left panel) and non-specific binding (centre panel; determined with 10 µM BU224) in 14 µm frontal cortex sections of the human brain (Braak stage 6). Standards (ARC 123B) represent a linear range of radioactivity. Immunohistochemistry for GFAP (right panel) shows spatial distribution of an astrocytic marker. Magnifications of ³H-BU99008 binding and GFAP staining are shown in the panels. Solid red arrows point to high-intensity ³H-BU99008 binding and corresponding areas of GFAP staining. E, F Common Aβ tracers do not displace ³H-BU99008 binding. Autoradiograph showing ³H-BU99008 total binding (left upper panel) and non-specific binding (left lower panel; determined with 10 µM BU224) in 14 µm frontal cortex sections of the human brain (Braak stage 2). Standards (ARC 123B) represent a linear range of radioactivity. Upper/lower right row shows binding of ³H-BU99008 in the presence of two commonly unlabelled PiB and Florbetaben, two commonly used Aβ tracers in ascending concentrations, showing no displacement. Quantitative analysis of specific binding (E) in the grey and white matter and in the presence of unlabelled PiB and Florbetaben (10–10000 nMol/L) in 14 µm frontal cortex sections (Braak stage 2). Quantification indicates that high-intensity ³H-BU99008 accumulation around plaques is likely not caused by binding to common Aβ-binding sites.