Dy506

The ipsilateral cortical tissues and serum were collected 1 day after dMCAo from rats. The tissue samples were homogenized in lysis buffer (PRO-PREPTM, iNtRON Biotechnology, Korea), centrifuged at 12 000 g for 30 min, and the supernatants were collected and stored at -80 °C. The cytokine (TNFα, IL-1β, and IL-6, IL-10) levels were quantified using commercial ELISA kits (DY510; DY501; DY506; DY522; R&D Systems Minneapolis, MN, USA) according to manufacturer's instructions. The cytokine levels were normalized to the total protein concentration of the sample.

Plasma protein levels of IL-6 were determined by a rat enzyme-linked immunosorbent assay (R&D Systems; DY506, Abingdon, Oxon, UK). All samples were analyzed in duplicate and measured at an optical density of 450 nm.

Hepatic tissues were homogenized with three volumes of buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.1% sodium dodecylsulfate (SDS), and 1% Triton X-100. The homogenates were centrifuged at 3000× g and 4 °C for 15 min. Then, the supernatants were collected for the subsequent analysis. Inflammatory cytokines, including IL-1β, IL-6, IL-10, and tumor necrosis factor-α, were detected using commercial enzyme-linked immunosorbent assay kits (dy501, dy506, dy522, and dy510; R&D Systems, Minneapolis, MN, USA).

Levels of IL-6, PGE2 and TNF-α in brain samples (supernatants) were determined with specific ELISA kits according to the manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA; catalog numbers DY506, SKGE004B and DY510, respectively). The detection limits of the assays were as follows: IL-6—125–8000 pg/mL, PGE2—39–2500 pg/mL, TNF-α—62.5–4000 pg/mL. Whenever the level of the tested constituent was less than the lower detection limit of the assay, results were marked as “undetectable” and given a value of zero.

All kidneys were homogenized in cold 5 mM sodium phosphate buffer. The homogenates were centrifuged at 12,000g for 15 min at 4 °C, and the supernatants were used to determine TNF-α, IL-6, hyaluronic acid, malondialdehyde (MDA), and protein carbonyl levels. The levels of these markers were expressed as per gram of protein (Bradford assay). To determine the oxidative stress and inflammatory cytokines levels, enzyme-linked immunosorbent assay (ELISA) kits were used. Tumor necrosis factor-α (TNF-α) (DY510, R&D system, Inc. Minneapolis, USA), interleukin-6 (IL-6) (DY506, R&D system, Inc. Minneapolis, USA), tissue MDA, and protein carbonyl were determined in homogenized tissue samples.

To examine the levels of proinflammatory cytokines in the striatal lysates, an equal amount of proteins (50 μg) were conducted to the ELISA of proinflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) (Cat. DY501, DY506, DY522, and DY510, respectively) (R & D Systems, Minneapolis, MN, the United States according to the manufacturer's instructions (Liew et al., 2019 (link)).