Anti foxp3 antibody

Blood obtained from the animals at the time of perfusion was collected in EDTA-tubes to avoid blood coagulation. 30µl of the blood were incubated with surface antibodies in PBS as indicated per spleen and lymph nodes for 15 min at RT. Cells were washed with flow FCSB and centrifuged at 300 g for 5 min at 4 °C. Red blood cells were then lysed and cells were fixed with Optilyse B (Beckmann and Coulter) for 10 min at RT. Fixative was washed with ddH20 to finish red blood lysis for 15 min and centrifuged at 300 g for 5min at 4 °C. Then, cells were resuspended in 100 µL Fix & Perm B (ThermoFisher Scientific) with an anti-Foxp3 antibody (1:100; eBioscience, clone FJK-16S) overnight at 4 °C. Cells were then washed with FCSB and centrifuged at 300 g and 4 °C for 5 min. Final pellet was resuspended, data were acquired on a FACSCanto II and analyzed using FlowJo software version 9.0 (BD). To calculate cell numbers, singlets were identified by FSC-H versus FSC-A and viable cells gated on CD4 expression, and subsequently CD25⁺ and Foxp3⁺ and CD25⁺Foxp3⁺ cells.

To evaluate BMDC maturation and inhibitory phenotype, BMDCs were stained with fluorophore-tagged monoclonal antibodies against mouse CD11c (N418), mouse CD40 (3/23), mouse CD80 (16-10A1), mouse CD86 (CL-1), mouse I-A/I-E (MHC class II) (M5/114.152), PD-L1 (MIH7), PD-L2 (TY25), CD206 (C068C2), and dectin-1 (RH1). For T cell assay, LN cells and splenocytes were stained with fluorophore-tagged monoclonal antibodies against mouse mouse CD3 (145-2C11), CD4 (GK1.5), mouse CD25 (PC61), and mouse/human FoxP3 (FJK-16s). Prior to staining with specific antibodies, the cells were incubated with Fc block (purified anti-mouse CD16/32) to reduce non-specific binding. All antibodies were obtained from BioLegend (San Diego, CA, USA) except for the anti-FoxP3 antibody, which was obtained from eBioscience (San Diego, CA, USA). The stained cells were assessed by flow cytometry (CytoFLEX, Beckman Coulter, San Diego, CA, USA) and the data were analyzed using Kaluza Flow Analysis Software (Beckman Coulter). For flow cytometry acquisition, the live cells of all samples were acquired at 20,000 cells/sample. Accordingly, the same electronic gate was applied to all sample for flow cytometry acquisition and analyses.

The sections were stained immunohistochemically with anti-Foxp3 antibody (eBioscience, San Diego, CA, USA), then detected by diaminobenzidine (DAB) staining.