Pe conjugated anti il 17 antibodies

Spleen tissue sections 7 µm in thickness were used for immunostaining. To analyze populations of Th17 cells, we used fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and PE-conjugated anti-IL-17 antibodies (eBioscience, San Diego, CA, USA). Stained sections were observed with a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at × 400 magnification. Positive cells were counted, and values were expressed as means ± standard deviations.

Isolated CD4⁺ T cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 800 ng/mL ionomycin (Sigma-Aldrich) for 5 h, with Protein Transport Inhibitor (Invitrogen) added for the final 2 h. An Intracellular Staining kit (Life Technologies) and phycoerythrin (PE)-conjugated anti-IL-17 antibodies (eBioscience) were used following the manufacturer’s instructions. Foxp3 was stained by using a Foxp3 Staining kit (Invitrogen) including fluorescein isothiocyanate (FITC)-conjugated anti-Foxp3 according to the manufacturer’s instructions. For surface staining, PerCP-Cy5.5-conjugated anti-CD4 antibodies and FITC-conjugated anti-CD45 antibodies from eBioscience were used. The analysis was performed by using a FlowSight system (Amnis).