Goat anti mouse igg hrp antibody

Immunohistochemical staining was performed on 5 µm sections of paraffin-embedded tissue specimens. The sections were deparaffinized in xylene and rehydrated using a graded ethanol rinse series (50, 75, 85 and 95%). Masked epitope retrieval was performed by heating the sections in a microwave oven in 0.01 M sodium citrate buffer (pH 6.0) for 20 min at 35°C. Endogenous peroxidase activity was terminated by incubation in 3% H₂O₂ for 20 min at room temperature. The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min. The antibody-antigen complexes were visualized using DAB and counterstained with hematoxylin at room temperature for 5 min. Finally, the sections were dehydrated in ethanol (50, 75, 85 and 95% series), cleared in xylene, and examined using microscope (magnification, ×400; BX-51; Nikon). The number of CD160 positive cells in one view were counted and this was repeated 3 times. In all staining procedures, the positive controls showed clear staining, whereas there was no staining in the negative controls.

Expression of recombinant fusion proteins was analyzed using SDS-PAGE and confirmed by Western blotting. A 12% gel was used to visualize the recombinant protein via SDS-PAGE performance (15 (link)). Staining of the gels was performed with Coomassie Brilliant Blue G-250 following Bio-Rad Mini PROTEAN electrophoresis (Bio Rad, USA). For Western blotting, the proteins were separated by 12% gel and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membrane. After electrotransfring, PVDF membranes were blocked by BSA 2% (overnight at 4 °C). For identification of Fc-tag recombinant protein (CFP10:Fcγ2a), incubation of membranes was done for probing with goat anti-mouse IgG-HRP antibody (Santa Cruz, USA) at a dilution of 1:10,000 for 1 hr at room temperature. Also, in order to identify CFP10:His fusion protein, PVDF membranes were incubated for an hr at room temperature with His-probe (H3) HRP antibody (Santa Cruz, USA) at a dilution of 1:5000. Finally, the specific recombinant proteins were detected by enhanced chemiluminescence detection system (ECL).

Proteins from rat liver were extracted using the Bradford Protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Total protein (50 μg) was separated by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After electrophoresis, separated proteins were transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Triton X-100 (TBST) for 1 h at room temperature. The membranes were probed with mouse monoclonal anti-IGF-1 antibody (catalog No. sc-74116, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. After washing, the membranes were incubated with goat-anti-mouse IgG-HRP antibody (catalog No. sc-2005, Santa Cruz Biotechnology) for 1 h at room temperature as previously described.¹⁹ (link) Protein expression of IGF-1 was detected by an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Little Chalfont, UK) and visualized by a Fusion FX Image system (Vilber Lourmat, Torcy, France). All other chemicals used were of analytical grade, and were purchased from the Sigma–Aldrich Chemical Co.