42 Chapter 2 Table 2. Antibodies for immunohistochemistry Antigen Source Antibody # Species Dilution TSPO (PBR) Abcam AB109497 Rabbit 1:750 PLP Bio-Rad MCA839G Mouse 1:3000 HLA-DR (LN3) Biolegend 327011 Mouse 1:1000 Vimentin Homemade N/A Mouse 1:6000 GFAP Millipore AB5541 Chicken 1:1000 Olig2 Millipore AB9610 Rabbit 1:500 CD3 Agilent A0452 Rabbit 1:1500 CD20 Agilent M0755 Mouse 1:100 TMEM119 Merck HPA051870 Rabbit 1:250 P2RY12 ANASPEC AS55042A Rabbit 1:200 CD40 Bio-Rad MCA1590 Mouse 1:50 CD206 BD Biosciences 555953 Mouse 1:50 S100β Merck S2532 Mouse 1:200 GLUT-1 Merck AB1783 Guinea pig 1:100 TSPO = translocator protein, PLP = proteolipid protein, HLA-DR = human leukocyte antigen - DR, CD = Cluster of differentiation, GFAP = Glia fibrillary acidic protein, TMEM119 = transmembrane protein 119, P2RY12 = purinergic receptor P2Y12, GLUT-1 = glucose transporter Image and statistical analyses Images of each lesion type and randomly sampled areas of normal appearing white (NAWM) and grey matter (NAGM), and white and grey matter from controls were collected with a Leica DC500 microscope (Leica Microsystems, Heidelberg, Germany) at 200 x magnification. Fluorescent images were taken with a Leica TCS SP8 STED 3X confocal microscope. Pictures were analysed using ImageJ software and nuclei and positive cells were manually counted with the cell counter plugin (de Vos, University of Sheffield, UK). All nuclei except those within blood vessels were counted to determine the number of cells per field. Expression levels were analysed using TSPO+ pixels with a threshold of signal intensity that represented all DAB+ pixels per field. Inter-observer consistency shown with a correlation coefficient of 0.98. Data was analysed using GraphPad Prism 7.02. All data was tested for normality distribution with the Shapiro-Wilk normality test. Differences between lesion types were analysed using ANOVA or Kruskal-Wallis analyses. When positive, Dunnett’s post hoc analysis was performed to test the different groups to their respective NAWM or NAGM and control, corrected for multiple comparisons. Accordingly, white and grey matter from control cases were compared to NAWM and NAGMof multiple sclerosis tissue, respectively. Data was considered significant when P<0.05. Genotyping DNA extraction and genotyping were performed on snap frozen brain samples (LGC Group Ltd, Hoddesdon, UK). In brief, following DNA extraction, the SNP-specific KASPTM Assay mix and the universal KASPTM Master mix were added to the DNA samples and placed in a thermal cycler for a minimum of 35 cycles, producing an allele-specific fluorescent signal in accordance with primers specific to rs6971 and rs6972. Each allele-specific primer produces a unique tail sequence which is associated with a fluorescent resonant energy transfer cassette, labelled with a FAMTM dye, or HEXTM dye. Plates were read on a BMG PHERAStar plate reader (BMG Labtech GmbH, Germany). In-house Kraken software was used to automatically identify genotypes, which were verified by staff at the LGC facility.
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