Anti il 4 11b11

For the immunohistochemical analyses of the CNVs, isolated choroidal sheets with or without the retina were fixed in 4% paraformaldehyde and incubated with isolectin IB4 and primary antibodies including anti-IL-4 (11B11, Biolegend, San Diego, CA), anti-IL-4Rα (mIL4R-M1, BD Biosciences, Franklin Lakes, NJ), anti-Iba1 (FUJIFILM, Tokyo, Japan), anti-IL-13R (ab-79277, abcam, Cambridge, UK), anti-CD11b (M1/70, eBioscience, San Diego, CA), anti-CCL2 (2H5, Biolegend), anti-CCR2 (NBP1-48338R, Novus Biologicals, Centennial, CO), or anti-CD31 (390, Biolegend) with anti-Fc RII/RIII blocking antibody (R and D Systems, McKinley Place, NE) overnight at 4° C. Then, the choroidal sheets were rinsed and incubated with the secondary antibodies labeled with either Brilliant Violet 421, DyLight 488, Alexa Fluor 555, PE, Alexa Fluor 647, or DyLight 649, or control antibodies. DAPI and TO-PRO-3 iodide (T-3605, Molecular Probes, Eugene, OR) were used for nuclear staining. A confocal microscope (LSM730, Carl Zeiss, Oberkochen, Germany), or a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) was used for photographing the whole mounts.

Levels of IL-2, IFN-γ, IL-4, IL-6, and IL-17 in culture supernatants were quantified using a sandwich ELISA. The following pairs of capture and biotinylated detection rat anti-mouse mAbs were used: for IFN-γ, anti-IFN-γ (P4-6A2, Biolegend) and biotin-conjugated anti-IFN-γ (XMG1.2, Biolegend) Abs; for IL-2, anti-IL-2 (JES6-1A12, BD Biolegend) and biotin-conjugated anti-IL-2 (JES6-5H4, BD Biolegend) Abs; for IL-4, anti-IL-4 (11B11, Biolegend) and biotin-conjugated anti-IL-4 (BVD6-24G2, Biolegend) Abs; for IL-6, anti-IL-6 (MP5-20F3, BD Biosciences) and biotin-conjugated anti-IL-6 (MP5-32C11, BD Biosciences); for IL-17, anti-IL-17 Ab (TC11-18H10, BD biosciences) and biotin-conjugated anti-IL-17Ab (TC11-8H4, BD Biosciences). Capture Abs (2 μg/ml) were coated onto 96-well plates. After blocking with 0.5% BSA in PBS containing 0.05% Tween 20, the diluted samples and recombinant protein standards were incubated for 1 h at room temperature. Plates were then incubated with biotin-conjugated detection Abs (1 μg/ml) for 1 h at 37°C and reacted with streptavidin-conjugated horseradish peroxidase, followed by o-phenylenediamine. The reaction was terminated by addition of 0.5 M H₂SO₄. The absorbance at 490 nm was then measured, and a graph was created by analyzing three samples.

The following combination of fluorescently labeled primary antibodies against cell surface markers and intracellular cytokines were used: anti-CD3 (17A2), anti-CD4 (clones GK1.5 and RM4-5), anti-CD8a (53–6.7), anti-CD44 (IM7), anti-CD45R/B220 (RA3-6B2), anti-CD11c (N418), anti-CD103 (M290), anti-Ly6C (HK1.4), anti-I-A/I-E (M5/114.15.2), anti-IL17 (TC11-18H10.1), and anti-IL4 (11B11) from Biolegend, and anti-IFN-γ (XMG1.2) and anti-IL-13 (eBio13A) from eBioscience. Cells were analyzed using a LSRII flow cytometer running FACSDiva Software (BD Bioscience) and analyzed using FlowJo Software (Tree Star). Fixable Viability Dye eFluor780 (eBioscience), DAPI or 7-AAD Viability Staining Solution (Biolegend) were used according to the manufacturer’s instructions to exclude dead cells from analysis.

ILC3s were incubated for 2 h with αGalCer (100 ng/ml) or PBS, washed extensively and adoptively transferred into WT recipients (0.5–1 × 10⁵ cells/mouse). Recipient mice were euthanized 18–24 h following transfer and iNKT cell activation was measured by qPCR and flow cytometry.
For qPCR analyses, iNKT cells were sort‐purified from the spleen, mLN and SI‐LP of recipient mice as TCR‐β⁺CD1d‐tetramer⁺B220⁻ cells and RNA was extracted as described below.
For intracellular IFN‐γ and IL‐4 staining, single‐cell suspension were prepared and stained with B220, TCR‐β and CD1d‐tetramer prior to fixation and permeabilization with Fix/Perm buffer set (Biolegend). Fixed cells were stained with anti‐mouse IFN‐γ (XMG1.2, Biolegend) and anti‐IL‐4 (11B11, Biolegend). Dead cells were excluded from the analyses using a fixable viability stain (Biolegend).