Rat anti mouse il 6 antibody

Mice were sacrificed at 3 or 8 dpi, and the lung lobes were fixed, dehydrated and frozen. Freshly cut lung sections (5 μm thickness) placed on poly-L-lysine-coated slides were pretreated with 1:10 FcR blocking agent (Miltenyi Biotech, Gladbach, Germany) for 10 min and reacted with various antibodies as follows: goat anti-prosurfactant protein (proSP)-C antibody (Santa Cruz Biotech., Santa Cruz, CA), rabbit anti-phospho-Stat3 (Cell Signaling Technology), rabbit anti-phospho-Akt antibody (Cell Signaling Technology), rabbit anti-Iba 1 antibody (WAKO, Osaka, Japan), rat anti-mouse IL-6 antibody (Biolegend, San Diego, CA), rabbit anti-S100A4 antibody (Abcam, Cambridge, UK) or mouse anti-smooth muscle α-actin/SMA (Sigma-Aldrich, St. Louis, MO). After staining with each appropriate fluorescein-conjugated second antibody, the sections were observed under a fluorescence microscope (Axio Imager A2, Zeiss, Oberkochen, Germany). Nuclei were stained with 4’6’-diamino-2-phenylindole (DAPI).

ELISA and tissue section were employed to investigate the inflammation and liver damage of TAA-treated mice. The ELISA kit used for analysing plasma levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α), high mobility group protein B1 (HMGB-1), and interleukin-6 (IL-6) levels was purchased from BioLegend (San Diego, CA, USA). An antibody used for the immunohistochemical (IHC) detection of TNF-α expression in mouse liver was obtained from R&D Systems (Minneapolis, MN, USA). Rat anti mouse IL-6 antibody was purchased from BioLegend. (San Diego, CA). Goat anti mouse HMGB1 antibody was purchased from Chondrex. (Redmond, WA). DNA staining dye 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich. Tissue section, haematoxylin and eosin staining (H&E staining), and immunohistochemistry (IHC) were performed following previously described methods^{27 (link),40 (link)} using an IHC kit purchased from Thermo Fisher Scientific (Fremont, CA, USA). Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) was used to capture images. The specific staining signals (e.g. green channel of TNF staining) were quantified using ImageJ software (NIH, Bethesda, MD, USA).