Anti rat il6

The RCN9 rat colon carcinoma and H9c2 rat cardiomyoblast cell lines were procured from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ atmosphere. Berberine (BBR) (10 μM for 48 h unless stated otherwise, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was obtained from the listed manufacturers. For cytokine neutralization, antibodies against HMGB1 (anti-HMGB1, Biolegend, San Diego, CA, USA), TNFα (anti-rat TNFα, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and IL6 (anti-rat IL6, Abcam, Waltham, MA, USA) were utilized at a concentration of 0.5 μg/mL.

Paraffin-embedded sections were incubated at 4 °C with the following primary polyclonal antibodies: anti-rat-interleukin (IL) 1 beta (IL-1β) (Abcam, Cambridge, UK), anti-rat-IL-6 (Abcam), anti-rat-matrix metalloproteinase 13 (MMP-13) (Abcam), anti-rat-MCP-1 (Abcam), anti-rat-CCR2 (Novus Biologicals, Littleton, CO, USA) and anti-rat-IL-17 (Abcam). Specific first antibody was diluted by DAKO antibody diluent (Dako, Carpinteria, CA, USA) and incubated overnight. Then, the samples were incubated with the respective secondary biotinylated antibodies, followed by 30-min incubation with streptavidin-peroxidase complex. Reaction products were developed using 3, 3-diaminobenzidine chromogen (Dako). Numbers of positive cells were counted in high-power fields (magnification: 400×) with the aid of NIH ImageJ software and averaged for three randomly selected fields per tissue section.