Rat anti mouse f4 80 monoclonal antibody

Histological analysis was performed as previously described (26 (link), 27 (link)). Mice with peak EAE were anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M PBS. Lumbosacral spinal cords were immediately removed, post-fixed in 4% paraformaldehyde, and embedded in paraffin or OCT compound (Sakura Finetek Japan, Tokyo, Japan). Eight-micron-thick paraffin sections were stained with hematoxylin and eosin. Stained sections were analyzed on a NanoZoomer 2.0-RS slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). For immunofluorescence staining, the frozen sections (20 μm) were permeabilized with 0.1% Triton X-100 in PBS for 20 min, blocked with 5% bovine serum for 30 min, and then incubated overnight with rat anti-mouse F4/80 monoclonal antibody (CI:A3-1; 1:1,000, AbD Serotec, Raleigh, NC, USA), rabbit anti-mouse iNOS polyclonal antibodies (1:5,000, Merck Millipore), and goat anti-mouse Arginase-1 polyclonal antibodies (1:200, Proteintech) followed by incubation with Alexa Fluor 488 or 546-conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA). The stained cells were analyzed by examining six random fields per section using a deconvolution fluorescence microscope system (BZ-X800, Keyence, Osaka, Japan). The data were collected from three animals per group.

Liver tissue was fixed in 10 % (v/v) neutral buffered formalin solution, dehydrated with ethanol, cleared in xylene and embedded in paraffin. Then the paraffin-embedded blocks were cut into sections approximately 5 µm thick. After removal of paraffin with xylene, sections were stained with haematoxylin and eosin (Merck)^{(30 )} for morphological analysis, or were stained with Sirius red (Sigma-Aldrich) and counterstained with fast green (Wako) for determination of the area of fibrosis⁽³¹ (link)⁾. Immunohistochemical staining of hepatic monocytes/macrophages was performed by using sections of formalin-fixed, paraffin-embedded liver tissue as described previously⁽³² (link)^,33 (link)⁾. Briefly, after removal of paraffin, sections were incubated with a rat anti-mouse F4/80 monoclonal antibody (Serotec), followed by incubation with a biotinylated rabbit anti-rat IgG antibody (Dako) and peroxidase-conjugated streptavidin (Dako). Then colour was developed with diaminobenzidine tetrahydrochloride (Dojindo) and the sections were counterstained with haematoxylin. Images were acquired with an Olympus DP73 digital camera (Olympus IX-73; Olympus) under an inverted microscope (original magnification, ×140). The F4/80-positive area and Sirius red-positive area were quantified as a percentage of the total tissue area by using cellSens Dimension Olympus 1.15 software (Olympus).

Animal care and all experiments were conducted in accordance with the guideline of the Animal Committee of Tokyo Medical and Dental University. C57BL/6J mice were purchased from ORIENTAL YEAST Co., Ltd. (Tokyo, Japan). Mice were housed under a 12-hour light-dark cycle and allowed food and water ad libitum. λ-Carrageenan was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant mouse follistatin was purchased from R&D Systems Inc. (Minneapolis, MN). Rat anti-mouse monoclonal F4/80 antibody was purchased from AbD Serotec (Oxford, UK).