Alexa fluor 647 anti mouse f4 80

For the immunofluorescence analysis, liver tissues were fixed in 4% paraformaldehyde for 12 h at 4°C and then dehydrated in 30% sucrose solution. The tissues were then frozen in OCT (Sakura, Torrance, CA, United States) compound and sectioned into 20 μm slices using a freezing microtome (Leica, Germany). OCT was removed by washing three times in PBS, and the sections were immunostained with Alexa Fluor 647 anti-mouse F4/80 (BioLegend, Clone: BM8, Catalog: 123122), Alexa Fluor 647 anti-mouse CD11c (BioLegend, Clone: N418, Catalog: 117312) or Alexa Fluor 647 anti-mouse NK1.1 (BioLegend, Clone: PK136, Catalog: 108720) at 1:200 dilution. All the sections were imaged with Olympus IX83 confocal microscope outfitted with an UltraVIEW VoX 3D live cell imaging system (PerkinElmer). Images were analyzed with Image J software (National Institutes of Health).

Abemaciclib was purchased from MedChemExpress Co. Ltd. IMD‐0354 was purchased from Selleckchem Co. Ltd. MAL‐PEG₂₀₀₀‐NHS was purchased from Beijing Kaizheng Biotech Co. Ltd (Beijing, China). Poly (L‐lysine) (PLL, M_w = 3–7 w), 2,3‐Dimethylmaleic Anhydride (DMA), Cis‐aconitic acid anhydride (CA), 3,4,5,6‐Tetrahydrophthalic anhydride (TDA) and succinic anhydride (SA) were purchased from Sigma‐Aldrich Co. Ltd (American). NGR peptide (sequence: GCNGRCGC) was obtained from Shanghai Apeptide Co. Ltd (Shanghai, China). Polyamindoamine (PAMAM‐G5, M_w = 28 826) was purchased from CY dendrimer technology Co. Ltd (Weihai, China). Cell Cycle and Apoptosis Analysis Kit was the product of Beyotime Biotechnology Co. Ltd (Shanghai, China). Alexa Fluor 647 anti‐mouse F4/80, Brilliant Violet 421 anti‐mouse CD206, PerCP/Cy5.5 anti‐mouse F4/80, Alexa Fluor 488 anti‐mouse CD86, APC anti‐mouse CD206, APC anti‐mouse CD3, FITC anti‐mouse CD4, PE anti‐mouse CD8, PE anti‐mouse CD25, and Alexa Fluor 488 anti‐mouse Foxp3 were purchased from Biolegend. ELISA kits were obtained from Dakewei Co. Ltd (Nanjing, China). All other reagents and solvents were obtained from Sinopharm Co., Ltd (Shanghai, China) and Sigma‐Aldrich Co., Ltd (Shanghai, China).

Tumor tissue was snap frozen; 10 μM sections were fixed in acetone, permeabilized, and non-specific staining blocked. For Day 1 tissue, macrophages were visualized by co-staining with Alexa Fluor 647 anti-mouse F4/80 (BioLegend), CellTrace Violet, and anti-mouse fibroblast activation protein-α (FAP) (Thermo Fisher Scientific). Images were captured via confocal microscopy (Olympus FV12000). For calculation of macrophage infiltration, cells were stained with α-F4/80 (macrophage/red) and CellTrace Violet (tumor cells/blue). FIJI software was used to calculate the % of red pixels (macrophages)/% of blue pixels (tumor cells). At least 50 images were taken for each tumor, and 4–5 tumors were analyzed for each time point. The % of red pixels/% of blue pixels of each photograph per time point is shown in box plots. The boxes represent the interquartile range (25–75%) with a line at the median and whiskers representing the minimum and maximum values.