Quantitative Analysis of Neuroinflammation

Mice were perfused with Ca²⁺/Mg²⁺ free Tyrode’s solution followed by 4% paraformaldehyde with 0.4% picric acid in 0.16 M phosphate buffer. The brains were dissected, postfixed for 2 hours, and equilibrated with 10% sucrose. Brains were frozen and cryo-sectioned to obtain 14 (for the striatum) or 20 (for the midbrain) μm thick sections. After 1 hour in blocking solution (PBS+ 0.3% TrItox X-100+ 1% BSA), the tissue sections were immunolabelled overnight with primary antibodies against TH (1:500, Pel-Freez; 1:1000, Chemicon), IBA1 (1:1000, Wako), GFAP (1:1000, Abcam) and CD45 (1:100, Serotec). For fluorescent staining, Cy3- (1:400, Jackson Biolabs), Alexa 546- (1:400, Life Technologies) and Alexa 633- conjugated secondary antibodies (1:400, Life Technologies) were used. Confocal images were acquired by sequential scanning using a LSM800 or LSM880 microscope (Zeiss). Relative intensity of mitoYFP-labelled mitochondria in the striatum and IBA1, CD45 and GFAP immunoreactivities in the midbrain were quantified using Fiji software. Confocal images were thresholded and total intensity was measured using automatic particle counting.
Live microscopy on sorted cells was performed as previously described [43 (link)].