Oct medium

After PET imaging, animals were sacrificed and tissue in the region of LPS/PBS injections were dissected, placed in 4% paraformaldehyde for overnight incubation, and transferred to 30% sucrose solution overnight. Subsequently they were embedded in O.C.T. medium (Fischer Scientific, Waltham, MA, USA) and frozen overnight at −80 degrees. Samples were sectioned on a cryostat at 5–10-micron sections.
Anti-Granzyme B (Cat #4059, Abcam, Cambridge, MA, USA) immunofluorescence was performed according to a standard protocol [21 (link)] using Anti-Granzyme B primary antibody 1:100 and a secondary goat anti-rabbit antibody 1:1000 (Thermo Fisher, Waltham, MA, USA). Slides were imaged on an Echo Revolve 332 microscope (Echo) at 4×, 10×, and 40× magnification.

RNAscope was used as previously described (Meirsman et al., 2016a (link)). Mice (n = 3 D1R-CTL; n = 3 D1R-Gpr88) were killed by cervical dislocation and fresh brains were extracted and embedded in optimal cutting temperature (OCT) medium (Thermo Scientific) frozen and kept at –80°C. Frozen brains were coronally sliced into 20-µm serial sections by using cryostat (CM3050 Leica), placed in superfrost slides (Thermo Scientific) and kept at –80°C until processing. In situ hybridizations were performed using the RNAscope Multiplex Fluorescent Assay. GPR88 and D1R probes were alternatively coupled to FITC or TRITC while D2R probes were coupled with Cy5.

The frozen brains were embedded in optimal cutting temperature (OCT) medium (Fischer Scientific, Whitby, ON) in blocks that included tissue from every behavioral group. Coronal sections (20 μm thick) were obtained from each block and collected on slides treated with 3-aminopropyltriethoxysilane (Sigma-Aldrich, Oakville, ON) and stored at −80°C. Once thawed, sections were hybridized for 18 hours with full-length Arc riboprobes generated using MAXIscript kits (Ambion, Austin, TX) and digoxigenin-UTP labelling mix (Roche, Indianapolis, IN). Slides were subsequently incubated with anti-digoxigenin-peroxidase (1 : 400; Roche) for 2 h at RT, followed by Cy3 for 30 min at RT (1 : 50; PerkinElmer, Waltham, MA) and DAPI for 30 min at RT (Sigma-Aldrich) to stain nuclei. An Olympus FV1000 laser scanning confocal microscope obtained z-stacks (20 μm thickness, ~1.0 μm optical planes, and 0.7 μm interval between planes) in one random location in the DG_IP and two locations in the DG_SP, as well as from one random location in each of the proximal and distal regions of CA1, CA3a, and CA3c (see Figure 1). Neurons within the median of 20% of each z-stack were classified as Arc+ or Arc−. The total number of cells counted in each region is provided in Table 1.

Skin samples were collected into stabilizing reagents and appropriate fixatives, depending on the procedure. For gene and protein expression analysis, skin samples were placed in molecular biology reagent (RNA‐stay, A&A Biotechnology, Gdansk, Poland) and frozen at −80°C. For histology, skin biopsy samples were placed in 10% buffered formalin for 48 hours, and formalin‐fixed paraffin‐embedded (FFPE). For frozen sections, biopsy samples were fixed in 4% paraformaldehyde (4% PFA, POCH S.A., Gliwice, Poland) and embedded in OCT medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described previously.²⁹ Finally, the skin was placed in culture medium to initiate skin‐derived primary cell expansion. Moreover, the brains from 3 healthy horses were collected as reference material.