85 TSPO in neurodegeneration were then treated in 0.02% Triton X-100 (Sigma-Aldrich) before incubation with an Iridiumintercalator (Fluidigm) then washed in dH2O and air-dried. Image acquisition took place using a Hyperion Tissue Imager (Fluidigm) coupled to a Helios mass cytometer. The instrument was tuned using the manufacturer’s 3-Element Full Coverage Tuning Slide before the slides were loaded into the device. 4 500x500μm regions of interest within the grey matter were selected and then ablated using a laser at a frequency of 200Hz at a 1μm resolution. The data was stored as .mcd files compatible with MCD Viewer software (Fluidigm) then exported as TIFF files. Post-acquisition image processing using ImageJ (v1.53c) software allowed threshold correction and the despeckle function to reduce background noise. The data was opened with HistoCAT (BodenmillerGroup) to quantify the signal of each Ln-channel and exported as .csv files. Multiplex immunofluorescence To immunophenotype microglia/macrophages expressing TSPO in the marmoset CNS, a multi-color multiplex immunofluorescence panel was used to stain for Iba1, PLP, and TSPO. Deparaffinised sections were washed twice in PBS supplemented with 1 mg/ml BSA (PBS/ BSA), followed by two washes in distilled water. Antigen retrieval was performed by boiling the slide in 10mM citrate buffer (pH 6) for 10 min in an 800Wmicrowave at maximum power, after which they were allowed to cool for 30 min and washed twice in distilled water. To reduce nonspecific Fc receptor binding, the section was incubated in 250 μl of FcR blocker (Innovex Biosciences, cat. no. NB309) for 15 min at room temperature and washed twice in distilled water. To further reduce background, sections were coated with 250 μl Background Buster (Innovex Biosciences, cat. no. NB306) for 15 min at room temperature and washed twice in distilled water. Sections were incubated for 45 min at room temperature in a primary antibody cocktail containing antibodies diluted in PBS/BSA (Supplemental Table 1), washed in PBS/BSA and three changes of distilled water. They were then incubated for 45 min in a secondary antibody cocktail composed of secondary antibodies diluted in PBS/BSA containing DAPI (Invitrogen, cat. no. D1306, 100 ng/ml) (Supplemental Table 2), then washed once in PBS/BSA and twice in distilled water. To facilitate mounting, the sections were air-dried for 15 min at room temperature, sealed with a coverslip as described previously, and allowed to dry overnight prior to image acquisition. Imaging and statistical analyses Brightfield images were collected at 40x magnification using a Leica DC500 microscope (Leica Microsystems, Heidelberg, Germany, Japan), or a Leica DM6000 (Leica Microsystems, Heidelberg, Germany) or a Zeiss AxioImager.Z2 wide field scanning microscope for fluorescent images. For AD, APPNL-G-F, and TAUP301S tissue images were collected from the hippocampus. For ALS tissue, images of the ventral horn and the lateral column were obtained from cervical, thoracic, and lumbar spinal cord levels. For mouse EAE and SOD1G93A mice, images of grey and white matter of the spinal cord were collected per case. ImageJ software was used for picture analyses. Nuclei and stained cells were counted manually using the cell counter plugin (de Vos, University of Sheffield, UK), excluding nuclei at the rim of each picture and within blood vessels. To determine inter-observer variation 18 pictures were manually counted by 3 independent observers with a correlation coefficient of > 0.9. To determine single cell TSPO expression, IBA+ or GFAP+ cells were outlined manually using the imageJ using the ROI manager. Afterwards TSPO+ pixels were measured within IBA+ and GFAP+ ROIs per cell. Data were analyzed using GraphPad Prism 9.1.0 software. All data were tested for normal
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