Anti mouse nk1

Specimens of the harvested livers and tumor tissues were fixed in 4 % formalin followed by embedding in paraffin. Tissue sections (4 μm) were subjected to histopathology (H&E staining), immunohistochemical (IHC) staining and in situ apoptosis detection by TUNEL assay. For IHC, after dewaxing, antigen retrieval and endogenous peroxidase blocking steps the sections were subjected to staining procedures with purified monoclonal anti-mouse CD11b (BD PharMingen, San Jose, CA); anti-mouse NK1.1 (Biolegend, San Diego, CA); and anti-rabbit CD31 (Abcam, Cambrige, MA) primary antibodies, respectively. Subsequently, biotinylated goat anti-mouse or anti-rabbit immunoglobulins as secondary antibodies and streptavidin peroxidase complex reagent were applied. The visualization signals were developed with diaminobenzidine (DAB) chromogen substrate (DAKO, Carpinteria, CA and the slides were counterstained with Meyer’s hematoxylin and dehydrated through a series of ethanol and xylenes. In addition, intratumor microvessel density was microscopically counted in absolute values as previously described [29 (link), 34 (link)]. Finally, for detection of in situ apoptosis in the tumor tissues, TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling) assay was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals) as described previously [34 (link)].

ILC1s were isolated from the spleens of C57BL/6 mice by combining magnetic beads and flow sorting. Mouse spleen cells were used to generated single-cell splenocyte suspensions[24 ]. The obtained cells were incubated with biotin-conjugated antibodies (anti-CD3ε, anti-CD45R, anti-Gr-1, anti-CD11c, anti-CD11b, anti-Ter119, anti-TCR-αβ, and anti-FCεRI; Miltenyi, Belgish, Germany) to enrich lineage-negative cells following the Miltenyi magnetic bead cell isolation protocol. Enriched lineage-negative single cell suspensions were stained with an anti-mouse lineage cocktail (BioLegend, 145-2C11, RB6-8C5, RA3-6B2, Ter-119, and M1/70), anti-mouse CD127 (BioLegend, A7R34), anti-mouse NK1.1 (BioLegend, PK136), and anti-mouse NKp46 (BioLegend, 29A1.4) for flow cytometry sorting (BD FACS Melody) of lineage⁻ CD127⁺ NK1.1⁺ NKp46⁺ cells (which were considered ILC1s); these cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 0.5 ng/ml IL-7 (Gibco, PMC0071).

To evaluate which immune cells were required to confer the observed antitumor effect, one day before tumor resection and hydrogel implantation, NK cells and CD8⁺ T cells in mice were depleted by intraperitoneal injection of depleting antibodies (200 μg/mouse) every 3 days for five times in total. The antibodies used for depletion were anti-mouse NK-1.1 (catalog no. 108760; Biolegend) and anti-mouse CD8α (catalog no. 100764; Biolegend).