Mouse rat igf 1 immunoassay kit

Plasma GH and IGF-1 were analyzed by a Rat GH ELISA kit (Shibayagi, Co, Ltd., Gunma, Japan) and Mouse/Rat IGF-1 Immunoassay kit (R&D systems, Minneapolis, MN), respectively. For liver tissue content of IGF-1, livers were lysed with PBS containing the Complete Proteinase Inhibitor Cocktail (Roche). Lysates were subjected to centrifugation to remove debris, the protein concentrations in each supernatant were measured, and then supernatants were used for ELISA.

Hormone levels were measured using serum derived from blood obtained from decapitated rats. Serum GH and IGF-1 were analyzed using a Rat GH ELISA kit (Shibayagi, Co, Ltd., Gunma, Japan) and a Mouse/Rat IGF-1 Immunoassay kit (R&D Systems, Minneapolis, MN), respectively. Insulin was measured using a rat insulin EIA kit (Macrodia AB, Uppsala, Sweden).

To analyze protein levels of various analytes, enzyme‐linked immunosorbent assays (ELISA) and Bio‐Plex assays were conducted using sera or protein extracted from TA muscle tissue. To isolate sera, whole blood was collected by cardiac puncture and allowed to clot on ice for 30 min before centrifugation. Total protein was extracted from approximately 50 mg of TA muscle tissue (midbelly) using a Bio‐Plex Cell Lysis Kit (Bio‐Rad; Hercules, CA). A protease inhibitor cocktail (Sigma‐Aldrich; St. Louis, MO) was added to sera and protein extracts before storage at −80°C. Immediately prior to analysis, protein was quantified using the bicinchoninic acid (BCA) assay (Pierce Biotechnology; Waltham, MA). High mobility group box 1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE) were both detected in serum by ELISA (Chondrex, Inc.; Redmond, WA and Abcam; Cambridge, UK, respectively). IGF‐1 levels were detected in sera and protein extracts using the mouse/rat IGF‐1 immunoassay kit as described by the manufacturer's protocol (R&D Systems; Minneapolis, MN). Tissue levels of select chemokines and cytokines, monocyte chemoattractrant protein‐1 (MCP‐1), tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and inerleukin‐10 (IL‐10) were detected using the BioPlex Pro rat cytokine multi‐plex immunoassay (Bio‐Rad) according to manufacturer's protocol.