Dp12 bx

BM biopsies obtained from orthopedic surgery were examined histopathologically. They were fixed in Oxford fixative (formaldehyde 40%, glacial acetic acid, sodium chloride, distilled water), routinely processed and embedded in paraffin wax. Sections of 3 μm thick were cut and stained with hematoxylin and eosin. The following antibodies were used: CD3 (polyclonal Ab, dilution 1:50; Dako, Glostrup, Denmark), CD4 (clone 4B12, dilution 1:10; Novocastra, now part of Leica Microsystems, Wetzlar, Germany), Foxp3 (clone 22510, dilution 1:50; Abcam, Cambridge, UK). Staining was performed according to the manufacturer’s instructions. The EnVision Detection System (Dako Denmark A/S) was used for detection. Positive controls were performed using human tonsils. Negative (isotype) controls were performed using ready-to-use FLEX negative control mouse antibodies (cocktail of mouse IgG1, IgG2a, IgG2b, IgG3 and IgM; code nr IR750; Dako Denmark A/S). Samples were reviewed for expression of these proteins in a blinded study performed by a research scientist (MP-S). Appropriate cellular localization for immunostaining was membrane −CD3, −CD4 and nuclear −Foxp3. All photographs were taken using Olympus microscope cameras: DP72 Olympus BX63 and DP12 Olympus BX (Olympus, Tokyo, Japan).

Bone marrow samples obtained from six OA patients and six RA patients were examined histopathologically. BM samples were fixed in Oxford fixative (formaldehyde 40%, glacial acetic acid 2%, sodium chloride 8.7%, distilled water), routinely processed and embedded in paraffin wax. Sections 3 μm thick were cut and stained with haematoxylin and eosin. The following antibodies were then used for further staining: anti-CD8 (polyclonal Ab, dilution 1:50; Dako, Glostrup, Denmark), anti-CD4 (clone 4B12, dilution 1:10; Novocastra, now part of Leica Microsystems, Wetzlar, Germany) and anti-IL-17A (dilution 1:50; Santa Cruz). Staining was performed according to the manufacturer’s instructions. The EnVision Detection System (Dako Denmark A/S, Glostrup, Denmark) was used for detection. Positive controls were performed on human tonsils. Negative (isotype) controls were performed using ready-to-use FLEX Negative Control Mouse (cocktail of murine IgG1, IgG2a, IgG2b, IgG3 and IgM, code number IR750; Dako Denmark A/S). Samples were reviewed for expression of these proteins by a qualified histopathologist who was blinded to outcome. Appropriate cellular localization for immunostaining was membrane for CD8 and CD4 and cytoplasmatic for IL-17A. All photographs were taken using Olympus microscope cameras: DP72 Olympus BX63 and DP12 Olympus BX (Olympus, Tokyo, Japan).