Il21 r

At 48 h post-IL-21 and anti-IL-21R antibody injections, mice were deeply anesthetized using isoflurane and euthanized via intracardiac perfusion using 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) in 100 mM phosphate buffered saline (PBS; pH 7.4, Thermo Fisher Scientific, Waltham, MA) [1 (link)]. Brains were cryoprotected (10–30% sucrose gradient over 2–3 days) and sectioned coronally into 30 µm using a cryostat (Microm HM525, Germany). For each endpoint, 4 representative coronal brain sections of the amygdala and medial prefrontal cortex (mPFC) regions from each of the 4 animals per experimental group were selected at approximately 15 section intervals and stored in PBS. For the immunofluorescence labeling of microglial activation marker CD68, rat anti-mouse CD68 (1:500; AbD Serotec, Hercules, CA) primary antibody was used with Alexa Fluor 594 secondary antibody (1:500). Similarly, for the IL-21R staining with microglia (IBA-1), we use IL21-R (BioXcell, Lebanon, NH) and rabbit anti-IBA-1 (Wako Chemicals, Richmond, VA) antibodies. The secondary antibodies included donkey anti-mouse or anti-rabbit Alexa Fluor 488 or 568. Tissues were then DAPI nuclear counterstained and sealed in slow fade/gold antifade mounting medium (Life Technologies, Carlsbad, CA).