Fitc cd4 clone gk1.5

Spinal cords were dissected out from PBS perfused mice, placed in 4% paraformaldehyde (PFA, Sigma-Aldrich, Denmark) in PBS, then immersed in 30% Sucrose and frozen as described previously (Mony et al., 2014 (link)). Spinal cord sections (16 μm thick) were cut on a cryostat and stored at −80°C. In brief, sections were postfixed in 4% PFA, and after several washes in PBS and PBS containing 0.2% Triton-X100 (PBST), they were then incubated with blocking solution containing 3% Bovine serum albumin in PBST. Sections were stained with PE-CCR6 (clone 29-2L17, Biolegend) and FITC-CD4 (clone GK1.5). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen-Molecular Probes). Isotype-matched primary antibodies were used to control for non-specific staining. Images for CCR6 expression in CD4⁺ T cells were acquired using an Olympus BX51 microscope (Olympus, Denmark) connected to an Olympus DP71 digital camera, and combined using Adobe Photoshop CS version 8.0 to visualize double-labeled cells.

Flow cytometric analysis of enzymatically digested tumor tissue was essentially performed as described in reference 65 (link). MRC1+ macrophages were gated as PE-Cy7 CD11b⁺ (clone M1/70 BD Bioscience), PE F4/80⁺ (clone BM8; BioLegend), and FITC CD206 (MRC1)⁺ (clone C068C2; BioLegend). Granulocytic myeloid-derived suppressor cells (GMDSC) were gated as PE-Cy7 CD11b⁺, PE Ly6-G⁺ (clone 1A8; BD Bioscience), and PerCP-Cy5.5 Ly6-C^int (clone Hk1.4; eBioscience). T cells were characterized as APC CD3⁺ (clone 17A2; eBioscience) and either eFluor 450 CD8a⁻ (clone 53-6.7; eBioscience) FITC CD4⁺ (clone GK1.5; BioLegend) for T-helper cells or vice versa for cytotoxic T cells. DAPI was used as a viability stain.