Animals were daily monitored and sacrificed/perfused when showing cues of sickness (that is, dehydrated, lethargic, scruffy and/or hunched). For perfusion, mice were sacrificed by carbon dioxide (CO2) overdose and then transcardially injected with 20 ml ice-cold saline (0.90/00 NaCl), followed by 50 ml ice-cold 4% paraformaldehyde (PFA, pH 7.4; 4 °C). Brains were then extracted and post-fixed in PFA overnight at 4 °C. Thirty-six to forty hours before sectioning, brains were cryoprotected in a 30% sucrose solution. Frozen coronal sections (35 μm thickness) were cut with a cryostat (Leica) and were either collected onto positively charged slides or kept at −20 °C into an ethylenglycol-based cryoprotectant solution until being processed for immunostainings. Before performing cryosections, whole brain pictures were taken with a camera.
