Complete freund adjuvant (cfa)

For DC depletion, mice were injected with 500 ng DT (List Biological Laboratories) intraperitoneally at indicated time-points. All immunization procedures were performed in the rear footpad with 20 μL injection volume per footpad as described previously (14 (link)). Mice were immunized as indicated in each Fig. with 5 μg low-endotoxin OVA (Worthington Biochemical Corporation) together with 50 μg papain (P4762, Sigma) or 10 μL CFA (F5881, Sigma). For in vivo antigen-uptake experiments, OVA was labeled with Alexa Fluor 488 Protein Labeling Kit (A10235, Thermo Fisher) and injected (5 μg) in the rear foot pad together with 50 μg papain or with 10 μL CFA. The ipsilateral (dLN) and contralateral (ndLN) popliteal LNs were harvested at indicated time-points.

Complete Freund’s adjuvant (CFA) (Thermo Fisher, St. Louis, MO) was dissolved in a 1:1 ratio of saline:CFA prior to intraplantar (i.pl) injection. CB-13, AM6545, AM630 and rimonabant (all from Cayman Chemical Company, Ann Arbor, MI) were dissolved in vehicle consisting of 20% DMSO (Sigma Aldrich, St. Louis, MO), 8% ethanol, 8% Tween 80 (Thermo Fisher, St. Louis, MO) and 64% saline and administered via intraperitoneal (i.p.) injection in a volume of 5 mL/kg for behavioral studies. CB-13, AM630 and rimonabant were dissolved in DMSO (25 mg/mL) to create stocks that were frozen until use. On the day of the experiment, stocks were diluted with a volume of DMSO to achieve a concentration of 20% DMSO in the final solution. 95% ethanol was then added, the solution vortexed again, followed by Tween 80 and finally saline. For AM6545, the steps were the same as the other compounds used, but the solution was sonicated for approximately 45 minutes to get the compound to fully dissolve (when the solution was clear). For calcium imaging and electrophysiology studies, prostaglandin E2 (PGE2) (Thermo Fisher, St. Louis, MO) and CB-13 were dissolved in DMSO and diluted in external recording solution.

Oxycodone (generously gifted from the NIDA drug supply program) dissolved in 0.9% sterile saline was administered cumulatively at doses of 0.1, 0.32, 1.0, and 3.2 mg/kg body weight for Von Frey tests. Oxycodone was administered at a dose of 1 mg/kg for place escape avoidance paradigm and locomotor tests, whereas 0.15 mg/kg/infusion (inf) was used for self-administration. To ensure the appropriate dose was used for each animal injection, volumes were adjusted according to the rat’s body weight. For self-administration, oxycodone was delivered by syringe pumps, and injection volumes were adjusted daily according to body weight^{32 (link)}.
Cyclosporin (CYSP) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in saline containing 2% dimethyl sulfoxide at a dose of 4 μg/μl, which has been shown to inhibit calcineurin activity. Control animals received 2% dimethyl sulfoxide dissolved in saline (vehicle). CFA was purchased from Thermo Fisher Scientific (Waltham, MA) and dissolved in paraffin oil.