Routine fixation, decalcification, and paraffin embedding. Tissue sections from fore and hind paws were cut and stained with hematoxylin osin. All the slides were coded and evaluated by two blinded observers. The specimens were evaluated with regard to synovial hypertrophy, pannus formation, and cartilage/subchondral bone destruction. The degree of synovitis and destruction in every joint concerning finger/toes, wrists/ankles, elbows, and knees was assigned a score from 0 to 3. Occasionally one paw was missing in the histological sections, or embedded in such a way that it was impossible to evaluate the degree of synovitis and bone/cartilage destruction. Therefore, the total score per mouse was divided by the number of joints evaluated.permeabilised using the FoxP3/Transcription Factor Staining Buffer set from e(-)-Indolactam V web Biosciences and antibodies diluted in 16PERM buffer included in the kit. The antibodies were directly conjugated with fluorescein isothiocyanate (FITC), phycoerythin (PE), allophycocyanin (APC), V450 and APC-H7. Cells were stained as previously described and gating of cells was performed using fluorochrome minus one settings [35] and detected by FACSCanto IITM (BD Biosciences). Gating strategy for expression of IL10 was included in Supplemental Figure 2. Analysis with respect to the number of cells and mean flourescence intensity (geometric mean) were performed using FlowJo Software, Tree Star Inc. (Ashland, OR, USA).Determination of SOCS Expression in Lymph NodesRNA was isolated from lymph nodes using RNEasy mini kit (QIAGEN). The RNA quality was analysed using a Experion Bioanalyzer on a Experion RNA 1480666 StdSens chip (BioRad) prior to cDNA synthesis with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The gene expression of SOCS3 was analysed using primers FW 59-CTGGTACTGAGCCGACCTCTCT-39 and RV 59-CCGTTGACAGTCTTCCGACAA-39, the expression of SOCS1 was analysed using primers SOCS1 FW 59-AAGGAACTCAGGTAGTCACGGAGTA-39 RV 59-CCGTGGGTCGCGAGAAC-39. The gene expression was normalised to b-actin analysed with primers FW 59CTGACAGGATGCAGAAGGAGATTACT and RV 59GCCACCGATCCACACAGAGT. All reactions were amplified using Power SYBR green PCR Master 24272870 Mix (Applied Biosystems) and analysed on a Viia7 system (Applied Biosystems).Determination of mRNA Levels of IL-10 in Lymph NodesRNA was isolated from lymph nodes using RNEasy mini kit (Qiagen). The RNA quality was analysed using a Experion Bioanalyzer on a Experion RNA StdSens chip (Bio-Rad laboratories Inc., USA) prior to cDNA synthesis with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The expression of IL-10 gene was analysed using primers IL10 FW 59-CATTTGAATTCCCTGGGTGAGA and RV 59TGCTCCACTGCCTTGCTCTT. The gene expression was normalised to b-actin analysed with primers FW 59-CTGACAGGATGCAGAAGGAGATTACT and RV 59-GCCACCGATCCACACAGAGT. The reactions were amplified using Power SYBR green PCR Master Mix (Applied Biosystems) and analysed on a Viia7 system (Applied Biosystems).Determination of Cytokine ProductionBlood was centrifuged at 7000 g for 10 min. Serum was collected and stored at 220uC for further analysis. Previously prepared spleen cell culture were Deslorelin custom synthesis stimulated for 72 hours with denatured chicken CII 50 mg/ml, supernatants collected and kept in 220uC until further analysed. Two independent experiments were performed. In the first experiment serum protein levels of IL10 were measured in serum by Duoset ELISA (R D systems) according to the manufacturer’s reco.Routine fixation, decalcification, and paraffin embedding. Tissue sections from fore and hind paws were cut and stained with hematoxylin osin. All the slides were coded and evaluated by two blinded observers. The specimens were evaluated with regard to synovial hypertrophy, pannus formation, and cartilage/subchondral bone destruction. The degree of synovitis and destruction in every joint concerning finger/toes, wrists/ankles, elbows, and knees was assigned a score from 0 to 3. Occasionally one paw was missing in the histological sections, or embedded in such a way that it was impossible to evaluate the degree of synovitis and bone/cartilage destruction. Therefore, the total score per mouse was divided by the number of joints evaluated.permeabilised using the FoxP3/Transcription Factor Staining Buffer set from eBiosciences and antibodies diluted in 16PERM buffer included in the kit. The antibodies were directly conjugated with fluorescein isothiocyanate (FITC), phycoerythin (PE), allophycocyanin (APC), V450 and APC-H7. Cells were stained as previously described and gating of cells was performed using fluorochrome minus one settings [35] and detected by FACSCanto IITM (BD Biosciences). Gating strategy for expression of IL10 was included in Supplemental Figure 2. Analysis with respect to the number of cells and mean flourescence intensity (geometric mean) were performed using FlowJo Software, Tree Star Inc. (Ashland, OR, USA).Determination of SOCS Expression in Lymph NodesRNA was isolated from lymph nodes using RNEasy mini kit (QIAGEN). The RNA quality was analysed using a Experion Bioanalyzer on a Experion RNA 1480666 StdSens chip (BioRad) prior to cDNA synthesis with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The gene expression of SOCS3 was analysed using primers FW 59-CTGGTACTGAGCCGACCTCTCT-39 and RV 59-CCGTTGACAGTCTTCCGACAA-39, the expression of SOCS1 was analysed using primers SOCS1 FW 59-AAGGAACTCAGGTAGTCACGGAGTA-39 RV 59-CCGTGGGTCGCGAGAAC-39. The gene expression was normalised to b-actin analysed with primers FW 59CTGACAGGATGCAGAAGGAGATTACT and RV 59GCCACCGATCCACACAGAGT. All reactions were amplified using Power SYBR green PCR Master 24272870 Mix (Applied Biosystems) and analysed on a Viia7 system (Applied Biosystems).Determination of mRNA Levels of IL-10 in Lymph NodesRNA was isolated from lymph nodes using RNEasy mini kit (Qiagen). The RNA quality was analysed using a Experion Bioanalyzer on a Experion RNA StdSens chip (Bio-Rad laboratories Inc., USA) prior to cDNA synthesis with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The expression of IL-10 gene was analysed using primers IL10 FW 59-CATTTGAATTCCCTGGGTGAGA and RV 59TGCTCCACTGCCTTGCTCTT. The gene expression was normalised to b-actin analysed with primers FW 59-CTGACAGGATGCAGAAGGAGATTACT and RV 59-GCCACCGATCCACACAGAGT. The reactions were amplified using Power SYBR green PCR Master Mix (Applied Biosystems) and analysed on a Viia7 system (Applied Biosystems).Determination of Cytokine ProductionBlood was centrifuged at 7000 g for 10 min. Serum was collected and stored at 220uC for further analysis. Previously prepared spleen cell culture were stimulated for 72 hours with denatured chicken CII 50 mg/ml, supernatants collected and kept in 220uC until further analysed. Two independent experiments were performed. In the first experiment serum protein levels of IL10 were measured in serum by Duoset ELISA (R D systems) according to the manufacturer’s reco.
