Immunohistochemical Analysis of CX3CL1 and CX3CR1 in SSc-ILD Lung Tissues

Immunohistochemistry staining was performed on paraffin-embedded samples of 5 lung tissues from SSc-ILD patients using the VECTASTAIN ABC Elite kits (Vector Laboratories, Inc., Burlingame, CA, USA) as previously described [25 (link),26 (link)]. After deparaffinization, rehydration and antigen retrieval in sodium citrate buffer (pH 6.0), endogenous perioxidase was quenched with 3% hydrogen peroxide. Sections were incubated in appropriate blocking serum to minimize non-specific binding and endogenous biotin was blocked with an avidin/biotin blocking kit (Vector Laboratories). Slides were incubated overnight with primary antibody at 4°C. The primary antibodies for rabbit polyclonal anti-human CX₃CL1 (PA5 23062) and rabbit polyclonal anti-human CX₃CR1 (PA5 32713) were purchased from Thermo-Fischer Scientific (Grand Island, NY, USA). Primary antibody for goat polyclonal anti-human CD138 (AF 2780) was purchased from R&D systems. Specific labeling was detected with a species specific biotinylated secondary antibody and application of horseradish peroxidase-conjugated avidin-biotin followed by development with 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories). Stained slides were counterstained with hematoxylin, mounted and analyzed for cellular sources of specific chemokines and their receptors.