Additional file 9.
Immunohistochemical and immunofluorescence analysis of paw sections. At the end of the therapy, paws from different therapy groups were frozen in cryoembedding medium (Neg50; Thermo Scientific, Wohlen, Switzerland) and stored at -80°C for analysis. (a) Hematoxylin and eosin staining of healthy and inflamed paw. Paw cryosections (10 μm) were fixed in ice-cold acetone and stained with hematoxylin solution Gill No. 2 (Sigma Aldrich) and alcoholic eosin Y solution (Sigma Aldrich). 10× magnification; scale bars = 50 μm. (b) Immunofluorescence analysis of infiltrating cells. Cryosections (10 μm) were fixed in ice-cold acetone and immunofluorescence staining was performed using primary antibodies against the following antigens: rat anti-mouse CD45 (leukocytes, 1:200; BD Bioscience), rabbit anti-asialo GM1 (NK cells, 1:4,000; Wako Pure Chemical Industries, Tokyo, Japan), rat anti-mouse CD4 (CD4+ cells, 1:50; BioXCell, West Lebanon, NH, USA) and rat anti-mouse CD8 (CD8+ cells, 1:50; BioXCell). Donkey anti-rat AlexaFluor488 (1:200; Invitrogen) and goat anti-rabbit AlexaFluor488 (1:200; Invitrogen) were used as secondary antibodies for detection. Sections were mounted with fluorescent mounting medium (Dako, Baar, Switzerland) and analyzed with an Axioskop2 mot plus microscope (Zeiss, Feldbach, Switzerland). The following scoring system was used for semiquantitative analysis of infiltrating cells: 0 = negative, 1 = single areas of positive cells with weak to moderate staining intensity, 2 = single areas of positive cells with strong staining intensity or disseminated positivity with weak to moderate staining intensity, 3 = large areas of positive cells within the whole tissue section with moderate to strong staining intensity. 10× magnification; scale bar = 50 μm (for all images). (c) Results of semiquantitative analysis for the different infiltrating cells (n = 2, F8-muIL10 n = 1). Healthy*, mice were immunized but did not show any signs of inflammation.
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Doll et al. Arthritis Research & Therapy 2013 15:R138 doi:10.1186/ar4319