Pa5 23062

Protein analysis was performed for two genes (CX3CL1 and CX3CR1) using the following antibodies: polyclonal antibody PA5-23062 (CX3CL1) and polyclonal antibody PA5-19910 (CX3CR1), both from ThermoFisher Scientific Company (Waltham, MA). Reactions were automated in the BenchMark Ultra-VENTANA equipment or manual protocol [Novocastra Novolink kit, Leica Biosystems (Buffalo Grove, IL)]. In total, immunohistochemistry was evaluated in 34 cases: nine HB samples from the exome cohort, 17 additional HBs from the tissue microarray (37 (link)), and eight samples from the Texas Children's Hospital cohort, including a lung metastasis sample.

The viral envelop protein was detected using the pan-immune anti-flavivirus antibody (mouse IgG1/IgG2a, clone ATCC-HB-112 D1-4G2-4-15 hybridoma, ATCC, Wesel, Germany) and double stranded RNA (dsRNA) was detected with the anti-dsRNA J2 antibody (mouse IgG2a, Scicons, Budapest, Hungary). Cell markers were detected using anti-human antibodies against CX₃CL1 (rabbit polyclonal IgG, clone PA5-23062, Thermofischer Scientific, Waltham, MA), Iba-1 (rabbit polyclonal IgG, Wako, Richmond, VA) and fluorescent-labelled CX₃CR1-R-Phycoerythrin (rat IgG2b, clone 2A9-1, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).
The following secondary antibodies were used for staining: donkey anti-rabbit IgG fluorescent-labelled Alexa Fluor 647 (AF647) (Abcam, Cambridge, UK) and goat anti-mouse IgG2a fluorescent-labelled AF647 for flow cytometry, as well as goat anti-mouse IgG2a fluorescent-labelled AF488 and donkey anti-rabbit IgG fluorescent-labelled AF546 for confocal microscopy (all obtained from Thermofischer Scientific if not mentioned). Nuclei were stained using DAPI (Sigma-Aldrich, Saint Louis, MO). All concentrations for the use of the antibodies and stains were optimized in our laboratory.

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.