65 Activated microglia do not upregulate translocator protein Table 2. Antibodies for immunohistochemistry Antigen Host Source Antibody Dilution CD68 Mouse DAKO M0814 1:500 CD68 Rabbit Atlas Antibody HPA048982 1:4000 HLA-DP,-DQ,-DR Mouse DAKO M0775 1:400 HLA-DR (LN3) Mouse Biolegend 327011 1:750 IBA-1 Rabbit Wako 1919741 1:10.000 TSPO (PBR) Goat Novus Biologicals NB100-41398 1:750 P2RY12 Rabbit ANASPEC AS55042A 1:200 TMEM119 Rabbit Merck HPA051870 1:250 CD68 Mouse DAKO M0814 1:500 CD68 Rabbit Atlas Antibody HPA048982 1:4000 antibodies for 1 h at room temperature (Table 2). Autofluorescent background signal was reduced with Sudan black (0.1% in 70% EtOH) for 10 min after which sections were thoroughly rinsed. Nuclei were stained with 4,6-diami-dino-2-phenylindole (DAPI) and mounted with Fluormount (Invitrogen; #00-4959-52). For all fluorescent staining procedures negative controls in which either the primary or secondary antibodies were omitted were used to control the presence of background signal. Imaging and quantitative analyses Images of each lesion type were collected using a Leica DM6000 microscope (Leica Microsystems, Heidelberg, Germany) for fluorescent images. Images were obtained randomly in the white matter of controls, and from MS in normal appearing white matter (NAWM) and white matter lesions. Pictures were analysed using ImageJ software and stained cells were counted manually using the cell counter plugin (de Vos, University of Sheffield, UK). Cells were counted as single, double, or triple positive based, and co-localisation of markers was based on observation of overlapping fluorescent signal. To justify the accuracy of the counting, 18 pictures were manually counted by 3 independent observers (EN, EG, MM). The inter-observer consistency was evaluated, resulting in a correlation coefficient of > 0.90. For the Sholl analysis, microglia were manually traced using ImageJ software. Afterwards, the traced microglial masks were analysed using the Sholl Analysis Plugin14. Data was analysed using GraphPad Prism 8.2.1 software. All data was tested for normal distribution, using the Shapiro-Wilk normality test15. Significant differences between the lesions were tested using the one-way analysis of variance test (ANOVA)16, or the Kruskal-Wallis test, in case of non-normally distributed data. When positive, Dunnett’s post-hoc analysis was performed to analyse which groups differ significantly from their respective control or NAWM in MS tissue17. Data was considered significant when P < 0.05. Results Expression of microglia and macrophage markers in multiple sclerosis lesions The LN3 antibody (Table 2) was used to denote the expression of HLA-DR in human tissues18. To investigate whether the lack of expression of HLA-DR by some microglia or macrophages is due to the antibody specificity we used another antibody (CR3/43) targeting HLA-DR, DP and DQ. We compared expression in the white matter of controls (Fig. 1A) with non-neurological diseases, and in NAWM, active (Fig. 1B), chronic active (Fig. 1C), and inactive (Fig. 1D) MS lesions in post mortem tissue. Immunostaining of HLA-DR, DP, DQ and HLA-DR showed a
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