Hrp rabbit mouse

Liver samples were fixed in 10% formalin, embedded in paraffin and sectioned. After antigen retrieval procedure and endogenous peroxidase activity inhibition, sections were incubated with anti-von Willebrand Factor (1:400; Dako, Glostrup, Denmark) or anti-P-selectin (1:400; Biovision, Milpitas, CA) 1 h at room temperature. HRP-Rabbit/Mouse (Dako) secondary antibody was added. Colour development was induced by incubation with a DAB kit (Dako) and the sections were counterstained with hematoxylin. Sections were dehydrated and mounted. The specific staining was visualized and images were acquired using a microscope equipped with a digital camera and the assistance of Axiovision software. vWF relative volume was determined by point-counting morphometry on immunoperoxidase-stained sections, using a point grid to obtain the number of intercepts over vWF positive cells over the tissue. Six fields were counted in each liver. All measurements were performed by two independent blinded observers. The relative volume was calculated by dividing the number of points positive in sinusoidal areas by the total number of points over liver tissue.

Muscle biopsies were from the upper leg (m. quadriceps or m. vastus lateralis). Stainings were performed on acetone-fixed cryostat-sections with mouse-anti-human CD3 (clone UCHT1, Nova, USA), followed by HRP Rabbit/Mouse (Dako, UK), or with biotinylated rat-anti-human FOXP3, followed by R.T.U.Vectastain kit (Vector Laboratories, UK), and visualized using 3,3'-Diaminobenzidine. Please note that CD3 staining was used to stain T cells, since CD4 expression is not specific to T cells only. 1–4 infiltrated areas were analyzed per patient, depending on the number of infiltrated areas found in the section. Pictures of CD3+ and FOXP3+ stainings were taken of infiltrated areas at 20× magnification, on consecutive slides with the same infiltrated area, and for each picture 2 independent researchers counted all positive cells. The number of cells was adjusted to the number of pictures counted, to obtain an average number per analyzed area for each muscle section.
For immunofluorescent staining secondary antibodies used were: anti-mouse-alexa488, and streptavidin-alexa594 (Invitrogen, USA), pictures were taken at 20× magnification. Stainings of the paraffin embedded muscle sections for IL-17 and FOXP3 analyses were performed according to previously published methods [20] (link).