Microm hm550

The frozen midbrains were stored at −80 °C and sectioned at the level of the SN, which can be clearly identified by its dark color15 (link). With a Microm HM550 cryostat (Thermo Scientific, Dreieich, Germany) combined with a fixed knife holder (Leica Biosystems, Nußloch GmbH, Nußloch, Germany), slices were cut at −18 °C object temperature and −20 °C chamber temperature. The 5, 10 or 20 μm thick sections were placed on 1.0 PEN membrane glass slides that were optimized for LMD (Carl Zeiss Microscopy GmbH, Göttingen, Germany).

C57Bl/6 mice were euthanized at 7 days post-injury and transcardially perfused with PBS, followed by 4 % paraformaldehyde. Brains were removed, post-fixed, and cryoprotected in 30 % sucrose. Thirty-micrometer sections were cut on a cryostat (Microm HM550, Thermo Fisher Scientific). Brain sections were treated with blocking buffer (10 % goat serum/0.1 % BSA/0.01 % Triton X-100) for 1 h at RT and stained O/N at 4 °C with antibodies against CD45-FITC (1:200, eBioscience, San Diego, CA, USA), CCR2 (1:100, Abcam, Cambridge, MA, USA), CD11b (1:200, Millipore, Darmstadt, Germany), and F4/80 (1:200, AbD Serotec, Raleigh, NC). After washing with PBS, three times, secondary Abs (1:200; anti-Rabbit-Cys3; Jackson ImmunoLaboratories, West Grove, PA, USA, anti-Hamster rat-Cys3; Jackson ImmunoLaboratories) were added and incubated for 1 h at RT. Slides were washed with PBS for 5 min, three times, and then coverslipped with mounting media with DAPI (Prolong Gold with DAPI, Invitrogen). The stained brain tissue sections were photographed with a ×20 objective using the BIOREVO all-in-one fluorescence microscope (BZ-9000 Generation II, Keyence microscope), and positive signal was measured using BZ-9000 Generation II analyzer (Keyence).