Mouse monoclonal anti il6

Sections of blood vessels were fixed in 4% buffered formalin for 24 h and subsequently dehydrated in ascending concentrations of ethanol, cleared in xylene, and embedded in paraffin. Blocks were trimmed and 3 µm sections were processed for immunohistochemistry. Primary antibodies used were: mouse monoclonal anti-FGFR1, 1:400 dilution (Abcam, Cambridge, UK); rabbit polyclonal anti-FGFR3, 1:300 dilution (Abcam); rabbit polyclonal anti-Klotho, 1:100 dilution (Abcam); mouse monoclonal anti-IL6, 1:300 dilution (Santa Cruz Biotechnology Inc., Dallas, TX, USA); and rabbit monoclonal anti-TNFα, 1:100 dilution (Santa Cruz Biotechnology Inc.). For the quantification analysis, a total of five images of each slide that include intima and media layers were captured and processed with a high-resolution video camera (Sony, DF-W-X710, Kōnan, Japan) connected to a light microscope (Nikon Eclipse 50i). The areas of tissue stained by the antibodies were quantified by using ImageJ software (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, USA). Results are expressed in square microns.

Western blotting was carried out as previously described (Hu et al., 2011 (link); Liu et al., 2014 (link)). Briefly, brains were divided into left and right cerebral hemispheres, cerebellum, and brainstem. The cerebral hemispheres opposite the blood clots were homogenized and equal amounts of protein (50 μg) were loaded on a Tris-glycine gel, separated by electrophoresis, and transferred to a nitrocellulose membrane that was blocked with blocking solution followed by overnight incubation at 4°C with the following primary antibodies: rabbit polyclonal anti-TTP (1:200), mouse polyclonal anti-PP2A (1:500), rabbit polyclonal anti-TNF-α (1:1,000), and rabbit polyclonal anti-IL-8 (1:1,000) (all from Abcam): mouse monoclonal anti-IL-6 (1:200; Santa Cruz Biotechnology); rabbit polyclonal anti-B cell lymphoma (Bcl)-2 (1:500; Sigma–Aldrich); and mouse monoclonal anti-caspase-3 (1:500; Millipore, Temecula, CA, USA). Membranes were incubated with appropriate secondary antibodies (1:2,000; Santa Cruz Biotechnology) for 1 h at 21°C, and bands were detected with a chemiluminescence reagent kit (ECL Plus; Amersham Bioscience, Arlington Heights, IL, USA). Band intensity was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA). β-actin (1:2,000; Santa Cruz Biotechnology) was used as a loading control.