Elisa kits for tnf α and il 6

Lipopolysaccharide (LPS, Escherichia coli O55:B5) was purchased from Calbiochem. Antibodies for inducible nitric oxide synthase (iNOS) and β-actin were purchased from BD Transduction Lab and Novus Biologicals, respectively. Cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) were purchased from Abcam. Total STAT3 and phospho-STAT3 (p-STAT3) were purchased from Cell Signaling Technology. ELISA kits for TNFα and IL-6 were purchased from Invitrogen. 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-acetyl-S-farnesyl-L-cysteine (AFC) were purchased from Sigma-Aldrich. Poly-ornithine was purchased from BD Biosciences. Papain and DNase I were purchased from Worthington Biochemical. Fetal bovine serum was purchased from HyClone and heat-inactivated. Culture media and penicillin/streptomycin were purchased from Gibco, and other common chemicals were from Sigma-Aldrich, unless stated otherwise.

ELISA kits for TNF-α and IL-6 were obtained from Invitrogen, USA. Reagents for immunohistochemical analysis included primary antibodies against NeuN (1:500, monoclonal, clone A60; EMD Millipore, MA), GFAP (1:500, rabbit polyclonal; Boster Biological Technology, CA), DCX (1:200, goat polyclonal; Santa Cruz Biotechnology, TX), Iba1 (1:500, rabbit polyclonal; Wako, Japan), and parvalbumin (PV, 1:3000, rabbit polycolonal; Proteintech, IL). The fluorescence-labeled secondary antibodies FITC-conjugated donkey anti-mouse IgG for NeuN, Cy3-conjugated donkey anti-rabbit IgG for GFAP, Iba1, FITC-conjugated donkey anti-rabbit IgG for PV, and Cy3-conjugated donkey anti-goat IgG for DCX were obtained from Jackson ImmunoResearch Inc., PA.

Tissues were collected from male mice aged 10–11 weeks old. Brain and liver tissues were homogenized in phosphate buffer saline (PBS) at pH 7.2 and subjected to MDA analysis by reaction with thiobarbituric acid (TBA)^{35 (link)}. Blood sample was collected by cardiac excision from anesthetized mouse using a syringe pre-washed with 0.5% heparin in saline. After centrifuging the blood sample at 1500×g for 10 min at 4 °C, plasma was collected and subjected to reactive oxygen metabolite (ROM) analysis by reaction with N,N-dimethyl-p-phenylenediamine to quantitate tert-butylhydroperoxide (tBHP) equivalents^{36 (link)}. ELISA kits for TNF-α and IL-6 (Invitrogen, USA) were used for brain assays according to manufacturer’s instructions.