Apc conjugated anti cd25

Tissue cryosections (7 µm-thick) were fixed in methanol-acetone and stained with FITC-conjugated anti-CD4, APC-conjugated anti-CD25, PE-conjugated anti-IL-17, and -Foxp3 (eBioscience). After incubation at 4°C overnight, the stained sections were analysed using a Zeiss LSM 510 Meta microscope (Carl Zeiss, Oberkochen, Germany) at ×200 magnification.

Subsequent to isolation of splenic lymphocytes with Lymphocyte Gradient Separation medium (Mediatech Inc, Herndon, VA), lymphocytes were stimulated with 10 ng/mL PMA, 1 μg/mL ionomycin, and 10 μg/mL Brefeldin A for 5 hours. Surface staining was performed before permeabilization using perm/wash buffer (BD Biosciences, San Diego, CA). Permeabilized cells were subsequently incubated with intracellular-targeting antibodies. Efluor 450-conjugated anti-CD3, FITC-conjugated anti-CD4, Percp-Cy5.5-conjugated anti-CD8a, APC-conjugated anti-CD25, PE-conjugated anti-FOXP3, Percp-Cy5.5-conjugated IL-17, and PE-conjugated IFN-γ were purchased from eBioscience. The BD LSR II with BD FACSDiva software flow cytometry system and FlowLogic software (Inivai, VIC, Australia) were used to analyze data.

For immunostaining, 7 μm tissue sections of spleens were stained. To analyze the populations of T helper cells, we used Alexa 488 conjugated anti-CD4, PE-conjugated anti-IL-17, APC-conjugated anti-CD25, and PE-conjugated anti-Foxp3 antibodies (eBiosciences, San Diego, CA, USA). To analyze the populations of STAT, AMPK and mTOR, the samples were stained with Alexa 488 conjugated anti-CD4, PE-conjugated anti-phosphorylated STAT-3 tyrosine 705, PE-conjugated anti-phosphorylated STAT-3 tyrosine 727, anti-mTOR, anti-AMPK, and anti-FGF21 antibodies, and anti-rabbit IgG-PE secondary antibody. The nuclei were stained with 4′,6-diamidino-2-phenylindole. The stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at × 400 magnification. Positive cells were counted, and the numbers expressed as the mean±s.d.

For analyses of the phenotype and frequency of each cell subset, peridin chlorophyll protein (PerCP)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD45RO, fluorescein isothiocyanate (FITC)-conjugated anti-interferon (IFN)-γ, allophycocyanin (APC)-conjugated anti-IL-17a, PE-conjugated anti-IL-23R, FITC-conjugated anti-CD4, APC-conjugated anti-CD25, PE-conjugated anti-FoxP3, APC-conjugated anti-CD39 (all from eBioscience, American) and phycoerythrin cyanin-7 (PE-Cy7)-conjugated anti-CD45RA (BD Bioscience) were used. For intracellular IL-17a and IFN-γ staining, cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA) and 1 μg/mL ionomycin (both from Sigma-Aldrich) in the presence of 10 μg/mL GolgiStop (BD Bioscience) for 5 hours at 37 °C in a humidified 5% CO₂ incubator before staining. The cells were extracellularly stained, then treated with Cytofix/Cytoperm solution and Perm/Wash solution (BD Bioscience). For intranuclear FoxP3 staining, cells were first extracellularly stained and then treated with Fixation/Permeabilization and Permeabilization Buffer (FoxP3 Staining Buffer Set, eBioscience, American). The appropriate isotype controls were used in all the staining procedures, and the stained cells were processed with a BD FACS Calibur and analysed using FlowJo software, version 7.6.1.

Cells were stained with Percp-conjugated anti-CD4 Ab (BD Pharmingen), then stained with APC-conjugated anti-CD25, PE conjugated anti-Foxp3 and FITC-conjugated anti IL-17 (all from eBiosciences, San Diego, CA, USA), followed by fixation and permeabilization using the Buffer Set (BD Biosciences) according to the manufacturer's instructions. All samples were run on a FACSCalibur (BD Pharmingen), and data were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).

Spleen tissues were obtained on day 35 after first immunization. The tissue was stained using PE-conjugated anti-CD4, FITC-conjugated anti-forkhead box P3 (Foxp3), APC-conjugated anti-CD25, FITC-conjugated anti-IL-17, FITC-conjugated anti-pSTAT3 (Y705), and FITC-conjugated anti-pSTAT3 (S727) (all from eBiosciences, San Diego, CA, USA). Stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).

For confocal staining, 7 mM tissue sections of spleens were stained using PE-conjugated anti-CD4, FITC conjugated anti-IL-17, Foxp3 conjugated anti-FITC and APC conjugated anti-CD25 (all from eBiosciences). Stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at x400 magnification.