Anti nk1

Peripheral blood (50 μL) was collected and placed in 4 ml of mouse RBC lysis buffer (eBioscience). Splenic single-cell suspensions were prepared by mechanical dissociation of the spleen, followed by filtration, and lysis of the RBCs. Cells were stained with anti-mouse CD4, CD8α, CD19, CD3e, NK1.1, λσ-T, FoxP3, and granzyme B antibodies (eBiosciences). For granzyme B measurements, T cell activation was performed before Ab staining. Splenic single-cell suspensions were prepared and cultured in RPMI 1640 (containing 10% FBS) plus IL-2. T cells were activated for 7 days using a Dynabead Mouse T Activator CD3/CD28 kit. Antibodies used for flow cytometry in this study (anti-CD11c, anti-CD11b, anti-CD19, anti-NK1.1, anti-CD3, and anti-MHCII) were from eBioscience, and anti-TNF-α was from Becton-Dickinson. Single-cell suspensions were prepared with a Neural Tissue Dissociation Kit from Miltenyi Biotec. For CD11b and TNF-α analysis, cells were treated with Cell Stimulation Cocktail and a protein transport inhibitor (eBioscience) overnight, stained for surface CD11b, and fixed and permeabilized for TNF-α detection (using flow cytometry). TMEM 119 was used to stain microglia via ex vivo flow cytometry and by immunohistochemistry (34 (link)).

Carbon tetrachloride, corn oil, percoll, collagenase type I, DNase I and pronase were purchased from Sigma Aldrich. The anti-desmin and anti-αSMA antibodies were purchased from Abcam. For immunohistochemistry, the anti-αSMA antibody was purchased from DAKO. The anti-Rae1 and anti- NKG2D antibodies were purchased from R&D systems. NKT, NK, CD3, CD4 and CD8 T cells expression from liver MNCs and splenocytes were identified by using anti-NK1.1, anti-CD3, anti-CD4, anti-CD8, anti-NKG2D, and anti-CD8 antibodies using flow cytometry purchased from Ebiosciences. anti-CD3, anti-CD4 and anti-CD8 antibodies were purchased from Abdserotec for immunohistochemistry application.