Goat anti rabbit igg biotin

Primary antibodies: anti-alpha 1 adrenergic receptor (α1-ADR, Abcam, ab3462, Cambridge, UK); anti-beta 2 adrenergic receptor (β2-ADR, Abcam, ab36956); anti-beta-Arrestin-2 (β-Arrestin-2, Cell Signaling, Cambridge, UK, Clone: C16D9); anti-cyclic AMP-response element binding protein (CREB, Abcam, Clone: E306); anti-phospho-cyclic AMP-response element binding protein (pCREB, Abcam, Clone:E113); anti-extracellular regulated kinase 1/2 (ERK1/2, ThermoFisher Scientific, Waltham, MA, USA, Clone: K.913.4); anti-phospho-extracellular regulated kinase 1/2 (pERK1/2, Cell signaling, Clone: D13.14.4E; anti-G-protein-receptor-kinase-2 (GRK-2, Abcam, Clone: Y137); Interleukin-10-Phycoerythrin-conjugated (IL-10-PE, eBioscience, Frankfurt, Germany, Clone: JES5-16E3); anti-p38 mitogen activated kinase (p38 MAPK, Cell signaling, Clone: D13E1); anti-phospho-p38 mitogen activated kinase (pp38 MAPK, Cell Signaling, Clone: D3F9).
Secondary antibodies: goat anti-rabbit IgG biotin (Dako, Frankfurt, Germany; catalog number: E0432); goat anti-rabbit IgG-R-PE (Sigma-Aldrich, St. Louis, MI, USA, catalog number: P9537); Streptavidin-PE (eBioscience, ThermoFisher Scientific, catalog number: 12-4317-87).
Isotype controls: mouse IgG (Abcam, ab37355); rabbit IgG (Abcam, ab172730, Clone: EPR25A).

Primary murine hepatocytes were incubated with rabbit-polyclonal-anti SLC22A1 (GeneTex International Corporation, Irvine, CA, USA) as primary antibody after preincubation with hydrogen peroxide for blocking of endogenous peroxidase. Endogenous biotin was blocked with the Avidin-Biotin Blocking kit (Vector Laboratories, Burlingame, CA, USA) and contaminating proteins were inhibited by ROTI®-Immunoblock solution (ROTH, Karlsuhe, Germany). After incubation with the secondary antibody (goat anti-rabbit IgG-Biotin, 1:1000; DAKO Cytomation, Hamburg, Germany) the TSA™ Cyanine system (Perkin Elmer, Waltham, MA, USA) was added. For negative control the primary antibody was omitted. The images were evaluated under a fluorescence microscope (Olympus BX51, Olympus U-RFL-T).

Samples were immersed in 0.1 M phosphate-buffered saline (PBS) for 5 min. Kits of primary polyclonal antibodies against TNF-α (code; ab667, supplier; ABCAM^®, UK) and S100 protein (Ac651 supplier; DAKO^®, Glostrup, Denmark) were used, and all instructions by the manufacturers were followed. The slides were washed with PBS for 5 min, then placed for 1 h at room temperature with drops of goat anti-rabbit IgG-biotin (1:200; DAKO^®) as the secondary antibody. After further rinsing, 3,3-diaminobenzidine covered the sections, followed by immersion in nonionized water to stop the reaction, and then, with Mayer’s hematoxylin for 30 sec and washed with PBS [19 ]. Sections were examined using an Eclipse E200-LED light microscope (NIKON^®, Japan).