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Finally, the cells were washed twice and analysed by flow cytometry. Cells were analysed on a FacsCanto (BD Biosciences) as described in detail elsewhere. For quantitative evaluation, dead cells were excluded by 7-AAD (Sigma, Deisenhofen, Germany) staining, and dendritic cells were detected either by monoclonal antibody (mAb) phycoerythrin (PE)-labelled CD1a (T6RD1, IgG1, Beckman Coulter, Krefeld, Germany) or by Fluoresceinisothiocyanat (FITC)-labelled CD1a (HI149, BD Biosciences). Expression of TLR2 was detected by mAb TL2.1 (eBiosciences, San Diego, CA, USA). TLR4 was either detected by mAb HTA125 (eBiosciences) or PE-labelled mAb 76B357.1 (Biomol, Hamburg, Germany). FARP1 Allophycocyanin (APC)-conjugated goat anti-mouse (GaM/APC) or FITC-conjugated goat anti-mouse (GaM/FITC) from Jackson Laboratories (West Grove, PA, USA) were used as secondary antibodies in indirect staining protocols. Normal mouse serum for blocking purposes was obtained from Dianova (Hamburg, Germany), Obeticholic Acid and 7-amino-actinomycin-D (7-AAD) to exclude dead cells was from Sigma. T cells were detected by mAb APC-Cy7-labelled CD3 (SK-7, IgG1, BD Biosciences). Cytokines were stained by PE- or FITC-labelled mAb B27 (IFN-��, IgG1, BD Biosciences), 8D4-8 (IL-4, IgG1, BD Biosciences), rat antibody (rAb) JES3-9D7 (IL-10, IgG1, BD Biosciences), mAb ebio64DEC17 (IL17A, IgG1, eBiosciences) and mAb 1D11 (TGF-��1, IgG1, R&D Systems, Wiesbaden, Germany). MAb FITC- or PE-labelled MOPC-21 (IgG1, BD Biosciences) and rAb PE-labelled R35-95 were used as respective isotype controls. Acquired data were analysed by flowjo software (Tree Star Inc., Ashland, OR, USA). Serial paraffin wax sections (4?��m) of oral mucosal tissue from VBR (n?=?5), GT (n?=?5), SLR (n?=?5) and SK (n?=?5) were stained by mouse monoclonal antibody specific for CD3 (Clone PS1, Novocastra, Newcastle upon Tyne, UK) to detect T cells for 1?hour at room temperature after heat-mediated antigen retrieval. LSAB2 kit (DAKO, Hamburg, Germany) was used for secondary labelling with fast red chromogen. Immunohistochemical image data were analysed and quantified by stereological image processing software image-pro plus selleck products (Version 6.0; Media Cybernetics, Inc., Bethesda, MD, USA). Five random microscope fields were selected from each slide (��200), and the number of immunopositive cell/field was counted by means of the five fields. For statistical evaluation of statistical significances, the Wilcoxon (signed-rank) test was performed. These tests were realized using the spss 17.0 for Windows software (SPSS GmbH Software, an IBM Company, Munich, Germany). Results are shown as arithmetic mean?��?standard error of the mean (SEM); *P?
