Tissue extraction reagent

Lung sections (40–60 mg of each) were homogenized in 300 µl of Tissue Extraction Reagent (Invitrogen) containing the Halt™ Protease Inhibitor Cocktail (Invitrogen). After incubation on ice (15 min) and centrifugation (13,000 rpm, 4 °C), supernatants were collected and stored on ice. Protein quantification was performed using the Bradford reagent (Sigma-Aldrich), according to the manufacturer’s instructions. The amount of inflammatory mediators in the lung homogenates were measured in triplicate with the flow cytometry-based method using magnetic microspheres conjugated with monoclonal antibodies (Luminex xMAP^® Technology). Cytokine and Chemokine 9-Plex Porcine ProcartaPlex™ Panel 1 (Invitrogen) allowed for simultaneous detection of IFN-α, IFN-γ, IL-1β, IL-10, IL-12/IL-23p40, IL-4, IL-6, IL-8 (CXCL8), and TNF-α. All measurements were performed on a BioPlex 200 platform with HRF (Bio-Rad), according to the manufacturer's instructions. The data was analyzed using BioPlex Manager 6.0 software (BioRad). The obtained values were normalized using the protein concentrations of each sample. The results are reported as pg/mg protein.

Brains were rapidly extracted, and right and left hippocampi were dissected and pooled together in the same microcentrifuge tube containing RNALater (Invitrogen, AM7020) and kept in a −80 °C freezer until used. Tissue was homogenized using a TissueLyser II (Qiagen, Germantown, MD, USA) for 30 s at a speed of 30 Hz in 200–500 µL of ice-cold homogenization/extraction buffer (20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 µL/mL phosphatase inhibitor cocktail 2 (Sigma, Cat #P5726), 10 µL/mL phosphatase inhibitor cocktail 3 (Sigma, Cat #P0044), RNase inhibitor). Homogenized samples were aliquoted for RNA and protein isolation. The homogenized samples were centrifuged at 10,000 × g for 5 min at 4 °C and the supernatant was transferred to a new tube. The total RNA was prepared using a standardized protocol based on TRIzol reagent (Invitrogen). The amount of RNA was assessed by using Nanodrop equipment (DeNovix DS-11 + Spectrophotometer). The protein was lyzed in Tissue extraction reagent (Invitrogen, FNN0071) in a volume of 100 µl. The protein concentration was measured using a Precision Red, Advance Protein Assay Reagent #2 (Cytoskeleton, Inc. Denver, CO, USA) following the manufacturer’s instructions.

UCP2, active caspase-3, phospho-AKT, IL-1β, and IL-6 concentrations of the rat hippocampi from control, SE, and SE ASO were quantified by ELISA. Tissues were excised and immediately frozen at −80°C. For protein extraction, the tissues were sonicated on ice in tissue extraction reagent (Invitrogen) containing protease inhibitor cocktail (Roche, Indianapolis, IN). After centrifugation at 12,000 xg at 4°C for 20 min, the supernatant was assayed for uncoupling protein 2 (UCP2) (Rat Mitochondrial uncoupling protein 2 ELISA kit, Cusabio®, Wuhan, China), caspase-3 activity (Caspase-3/CPP32 colorimetric assay kit, Biovision, Milpitas, CA), phospho-AKT (AKT-pS473 ELISA kit, Abcam, Cambridge, UK), IL-1β, and IL-6 (rat IL-1β or rat IL-6 Quantikine; R&D Systems, Abingdon, United Kingdom).

Cytokine and chemokine levels were determined in ipsi- and contralateral paws of Cre^- and Cre⁺ Mcpt5-DTA mice 48 h after injection of 10 µl 12 mg/ml zymosan, using the Bio-Plex Pro mouse cytokine group I (Bio-Rad). Tissue samples of paws were dissected and frozen directly at −80°C until protein extraction. The tissue was lysed in 400 µl lysis buffer (1x Protease Inhibitor Cocktail (#11697498001, Roche) in Tissue Extraction Reagent (#FNN0071, Invitrogen). Samples were cut in small pieces and then sonicated twice at 60% power for 10 sec with an Ultrasonic Homogenizer (SONOPULS HD2070 MS73, Bandelin). Afterwards all samples were centrifuged for 10 min at 10,000 g and the supernatant harvested. The concentration of total protein in the samples was assessed by the bicinchoninic acid assay. All samples were diluted to a final protein concentration of 200–900 µg/ml, according to the kit requirements. The concentrations of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL12-p70, IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), TNF-α were measured with a Bioplex 200 (Bio-Rad). The concentration was then normalized to the total protein concentration of the respective sample and is shown as pg/mg protein.