Aprotinin

Brains were immediately extracted from the skull (N = 4–5/group) and the hippocampus dissected from each cerebral hemisphere. Each hippocampus was weighed and transferred into 300 μL ice‐cold lysis buffer (N‐PER Neuronal Protein Extraction Reagent, Thermo Scientific) containing sodium orthovanadate (0.5 mM), phenyl‐methylsulfonyl fluoride (PMSF, 1 mM), aprotinin (10 μg/mL), and leupeptin (1 μg/mL; Santa Cruz Biotechnology, Santa Cruz, California). Tissues were sonicated individually, centrifuged at 4 °C for 15 min at 13,200 rpm, and the supernatants were collected and diluted 1:5 with Dulbecco's phosphate‐buffered saline. The supernatants were acidified to pH 2.6 then neutralized to pH 7.6 to liberate the neurotrophic factors. Following neutralization, each supernatant was diluted 1:10 prior to loading onto uncoated ELISA plates (Biolegend Nunc MaxiSorp, catalog number 423501) along with their respective BDNF or GDNF standards. The BDNF and GDNF levels were assayed using E_max ImmunoAssay Systems from Promega following the manufacturer’s protocol (BDNF catalog number G7611, GDNF catalog number G7621). Measurements were performed at a wavelength of 450 μm on a microplate reader (BioTek Synergy Mx) and linear regression of the standard curve was used to derive the pg of each neurotrophic factor per gram of hippocampal tissue.

Histopaque, p-nitrophenyl valerate (pNPVa), and LPS (E. coli 055:B5) were purchased from Sigma (St. Louis, MO, USA). AIM V^® Serum Free Medium was purchased from Thermo-Fisher (Waltham, MA, USA). RIPA lysis buffer and protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; 4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF; bestatin; pepstatin A; leupeptin hemisulfate; and aprotinin) were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for Western blots (anti-CES1, anti-MAGL, β-actin, goat anti-rabbit, and goat anti-mouse) were purchased from Abcam (Cambridge, MA, USA). Authentic 2-AG, anandamide, AA, and its deuterated analog AA-d8 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Small molecules JZL184 and WWL113 were purchased from Sigma. CpG was purchased from InvivoGen (San Diego, CA, USA). Primary CES1 and MAGL antibodies for flow cytometry were purchased from Abcam. Fluorescence secondary antibodies for flow cytometry and antibodies against IL-6 used to neutralize IL-6 or perform ELISA were purchased from Biolegend (San Diego, CA, USA). For some of the experiments, monocyte-depleted (n = 1) and whole PBMCs (n = 5) were purchased from Astarte Biologics (Bothell, WA, USA).

To determine the impact of RZ on the hippocampal BDNF, mice receiving ADR or RZ were euthanized for 4 weeks after the initiation of RZ treatment, and BDNF ELISA was performed as described [34 (link)]. Brains were immediately extracted from the skull (N = 8 to 9 mice per group). The hippocampus was microdissected from each cerebral hemisphere, flash-frozen by immersing the cryo-vials in the liquid nitrogen, and stored at − 80 °C until assayed. Each hippocampus was weighed and transferred into 500 μl ice-cold lysis buffer (NPER, Neuronal Protein Extraction Reagent, ThermoScientific) containing sodium orthovanadate (0.5 mM, Santa Cruz), phenyl-methylsulfonyl fluoride (PMSF, 1 mM, Santa Cruz), aprotinin (10 μg/ml, Santa Cruz), and leupeptin (1 μg/ml; Santa Cruz). Tissues were then sonicated individually and centrifuged at 4 °C, and the supernatants were collected and diluted at 1:5 or 1:10 with ice-cold Dulbecco’s PBS (Gibco). The supernatants were acidified to pH 2.6 and then neutralized to pH 7.6. The BDNF levels were assayed using a commercially available ELISA kit (E-EL-M0203, Elabscience Biotechnology) and uncoated ELISA plates (Nunc MaxiSorp, Biolegend). The colorimetric measurements were performed at 450 nm wavelength using a microplate reader (BioTek SynergyMx).