Goat anti rabbit fitc

Cells were obtained from bone marrow and spleen of FVB mice (Charles River Laboratories) after euthanasia under anesthesia. Cells were stained with rabbit anti-CLCF-1 polyclonal serum (Santa Cruz Biotechnology NNT-1/BSF-3 (FL-225)) which was raised against a human CLCF-1 peptide and cross-reacts with mouse and rat protein. Normal rabbit IgG (catalog #: sc2027) was used as an isotype control. Detection was performed with goat-anti-rabbit-FITC (Pharmingen 554020). Fixation and permeabilization solution from eBioscience was used as recommended. The data were analyzed using Becton-Dickenson FACSCalibur and FlowJo single cell analysis software.

For the detection of immune cells, cytokines, and transcription factors, the following antibodies were used: CD4 (GK1.5; Biolegend, San Diego, CA, USA), CD45 (30-F11; Biolegend), CD25 (PC61; Biolegend), CD8 (53-6.7; Biolegend), CD11b (M1/70; Biolegend), CD3 (17A2; Biolegend), IFN-γ (XMG1.2; BD Biosciences), T-bet (04-46; BD Biosciences), RORγT (Q31-378; BD Biosciences), IL-17A (TC11-18H10; BD Biosciences), Foxp3 (150D; Biolegend), CD45.1 (A20; BD Biosciences), CD45.2 (104; BD Biosciences), goat antirabbit FITC (BD Biosciences), and rabbit antimouse dopamine D1 and D2 receptor (both from Merck, Darmstadt, Germany). For cell surface staining, cells were incubated with Fc Block (2.4G2; BD Biosciences) for 15 min prior to staining with fluorescently labelled antibodies for 20 min on ice. For intracellular staining, cells were fixed, permeabilized, and stained using Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s protocol. Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analyzed using Flowjo software (Treestar Inc., Ashland, OR, USA).

After washing twice with PBS and blocking with 10% sheep serum (Sigma-Aldrich, St. Louis, MO, United States) for 10 min, cell suspensions were incubated for 30 min at 4°C with polyclonal C17 antibody (Abcam, #184996, Cambridge, United Kingdom) as a primary antibody, diluted 1:1,000 in PBS. Following washing steps with PBS, cells were incubated with the secondary antibody (goat anti-rabbit FITC) (BD Biosciences, Franklin Lakes, NJ, United States) diluted 1:25 in PBS for another 30 min at 4°C in the dark. Cells were analyzed using the FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, United States). Data analysis was performed using the Kaluza software (Beckman Coulter, Brea, CA, United States).