Ab183685

The fresh tumor was fixed with 10% neutral buffered formalin before paraffin embedding. For histological analysis, 5 mm thick sections were cut and stained with hematoxylin and eosin. Paraffin-embedded sections were stained for NK cells with CD49b (1:100, 108913, BioLegend), B cells with B220 (0.5 µg/ml, ab10558, Abcam), macrophage with F4/80 (1:200, ab16911, Abcam), dendritic cells with CD11c (1:100, ab219799, Abcam), CD4 (1:1000, ab183685, Abcam) and CD8 (1:2000, ab217344, Abcam), as well as LAMP2 (1:500, ab18528, Abcam). A Goat anti Rabbit IgG polyclonal HRP conjugate was used as the secondary. Immunofluorescence staining was applied to assess the presence of tumor-infiltrating CD4 (1:1000, ab183685, Abcam) and CD8 (1:1000, ab217344, Abcam) T cells. Immunohistochemical slices were transformed into high-resolution digital images using Nano Zoomer S60 scanner (Hamamatsu), and image preprocessing and quantitative analysis were performed using NDP.View.2 software. The positive cells of CD49b, B220, F4/80, CD11c, CD4, and CD8 staining were counted after it annotated the tumor region (avoiding necrotic tumor areas). The LAMP2 staining was assessed using a semiquantitative score of 0 (no staining) to 3 (strong staining) by an investigator blinded to the groups.

The tumor tissue was fixed and embedded in paraffin. After that, the samples were cut into 3-µm-thick slices and stained with immunohistochemistry. The primary antibodies used in the study were as follows: anti-CCL11 (1:500; Abcam; ab203586), CD4 (1:200; Abcam; ab183685), and Foxp3 (1:2000; Abcam; ab75763). The primary antibody was incubated at 4℃ overnight. After washing with PBS, secondary antibody was added and incubated at room temperature for 1 h. With (DAB) color development, hematoxylin inhibits the nucleus. Finally, the staining quantity and intensity were comprehensively analyzed. The IHC results were evaluated using the intensity score. IHC scores were determined by multiplying the score for staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) with the score or positive area (0, less than 5%; 1.5–25%; 2.26–50%; 3.51–75%; 4, greater than 75%). Scores of 0 to 7 were considered as a low expression and scores of 8–12 as a high expression. The scoring criteria referred to the following literature (Shan et al. 2013 (link); Tuo et al. 2022 (link)).