Anti il6 antibody

MC3T3-E1 was treated with ALA and SFC for 1 h and then treated with LPS for 24 h. Brefeldin (3.0 μg/ml) were used to inhibit protein transport during culture. Cells were washed with PBS and treated with cell lysis buffer (5 mM EDTA, 10％ glycerol, 1％ Triton X-100, 0.1% SDS, 1％ NP-40) in PBS. Protein concentration in each of the lysates was measured with Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to be the same for each lysate. After mixing with sample buffer, it was heat denatured, were electrophoresed on a TGX Precast gel (Bio-Rad Laboratories). The proteins were transferred to a PVDF membrane, and blocked with PVDF Blocking Reagent (Toyobo Co. Ltd, Osaka, Japan). Membrane was then incubated with anti-IL6 antibody (1/2000 dilution; ProteinTech Group, Chicago, IL, USA). After washing 0.5% Tween-20 in PBS (PBS-T), the membrane was incubated with HRP-conjugated secondary antibody (Thermo Fishter Scientific, San Jose, CA). To confirm the amount of the loaded protein were equal, membrane was incubated with anti-β Actin antibody (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with LumiCube (Liponics, Tokyo, Japan).

After deparaffinization, the sections were incubated with 0.3％ H₂O₂ in methanol to quench the endogenous peroxidase activity and then treated with BLOCK ACE (DS PHARMA BIOMEDICAL, Osaka, Japan). The sections were incubated with anti-IL-6 antibody (ProteinTech Group). After washing with PBST, the sections were incubated with peroxidase-conjugated secondary antibody (Vector Laboratories, Burlingame, CA). After incubation, the sections were flooded with DAB solution (Vector Laboratories), counterstained with hematoxylin. Sections were mounted with Entellan (Merck) and observed with a microscope. Intensity levels were measured using Image J (colour deconvolution) (Schneider et al., 2012 (link)).