43 Translocator protein expression in multiple sclerosis Autoradiography Brain sections were prepared from frozen tissue blocks corresponding to the paraffinembedded tissue blocks described above, allowing direct comparison of the same lesion for pathology and autoradiography. Sections were cut at 10 µm and thaw-mounted on standard glass microscope slides (VWR International Ltd, PA). Slides were dried for 30-60 mins at room temperature and stored at-80˚C. At the time of use, tissue had been stored for a maximum of 36 days. Prior to autoradiography, sections were thawed at room temperature for 15 mins, washed for 20 mins in assay buffer (50mM Tris-HCl, pH 7.4 and incubated for 1 h in assay buffer containing the radioligand [3H]-PK11195 1-(2-chlorophenyl)-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide (Perkin Elmer, specific activity 82.7 Ci/mmol) and [3H]-PBR28 (N-[2-(methyloxy)phenyl]methyl-N-[4-(pheyloxy)-3-pyridinyl] acetamide (Tritec, UK, specific activity 81 Ci/mmol). The concentrations were 1 nMol/L for [3H]-PK11195 and 0.5 nMol/L for [3H]-PBR28 as measured by liquid scintillation counting (Beckman LS 6500, UK). Adjacent sections were incubated with the radioligand and 10 µMol/L PK11195 (Tocris, UK) to determine non-specific binding. Following incubation, sections were washed twice for 2 mins each in washing buffer (50 mM Tris-HCl, pH 7.4/4°C) on ice followed by 30 sec in icecold ultra-pure water. All slides were air dried for 15-20 mins and dehydrated for a minimum of 24 h in a sealed container in the presence of phosphorous pentoxide. Slides were then exposed to BAS-TR2040 imaging plates (Fuji Film, Japan) alongside [3H]-standards (American Radiolabelled Chemicals, USA) for 19 days. Imaging plates were scanned using a Typhoon FLA 7000 (GE, UK) and analysed using QuantityOne (BioRad, USA). Autoradiography and genotyping analysis Regions of interest were drawn around lesions, normal appearing white matter (NAWM), and normal appearing grey matter (NAGM) on total binding images using immunohistochemical staining of adjacent sections as a reference. Corresponding regions of interest were drawn on the non-specific binding images for white and grey matter separately to determine the individual nonspecific binding. A global background reading was obtained from a free area on the plate and subtracted from all other measurements. Radioligand signal from each area of non-specific binding was also subtracted from the corresponding ligand-bound section to eliminate non-specific signal. Radioactive concentrations were calculated from optical densities using a linear regression derived from the radioactive standards. Final values are expressed as fmol/mg tissue equivalent (TE). rs6971 causes a single amino acid substitution at position 147 of the TSPO, which has a substantial impact on the affinity with which PBR28 binds TSPO. The binding affinity of PBR28 for TSPO is reduced by a factor of approximately 50 in subjects with threonine at position 147 (termed “low affinity binders”) relative to subjects with alanine at position 147 (termed “high affinity binders”). Heterozygotes (termed “mixed affinity binders”) express both copies of TSPO (147Alanine and 147Threonine) and hence present both the high affinity and low affinity binding sites in roughly equal proportion. Therefore, for a given expression level of the TSPOprotein detected by immunohistochemistry, the [3H]-PBR28 radioligand binding signal will be genotype dependent, showing rank order high affinity binders > mixed affinity binders > low affinity binders. Including all 3 genotypes in a correlation of the [3H]PBR28 signal with immunohistochemistry would therefore artificially weaken the correlation, and hence for this analysis, only high affinity binders were used. This phenomenon is not relevant for [3H]PK11195 as the affinity of this radioligand for TSPO is insensitive to the 147Threonine/Alanine substitution.
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