Gb13064 1

Mice were sacrificed on day 10 after CAR-T/M-THP1 and hMSC injection, and tumor samples were fixed with formalin and embedded in paraffin. Tumor tissues were examined by immunohistochemistry staining as previously described (Jiang et al., 2018 (link)). Briefly, the sections were exposed to 3% H₂O₂ in methanol after deparaffinization and rehydration and then blocked with 1% BSA for 30 min at room temperature. After blocking, the sections were incubated with primary antibodies (all from Servicebio Technology Co., China) for IL-1β (GB11113), CD4⁺ (GB13064-1), CD8⁺ (GB13068), and FOXP3 (GB11093) overnight at 4°C, followed by incubation with peroxidase-conjugated secondary antibodies. IL-1β+ cells were quantified by measuring the number of stained cells.

In short, samples were fixed in 4% paraformaldehyde, and embedded in paraffin for hematoxylin-eosin staining (H&E) or immunohistochemical analysis. Samples were incubated with CD4 (1:50, GB13064-1, Servicebio), CD8 (1:200, GB11068-1, Servicebio), and CD68 (1:200, GB14043, Servicebio) at 4 °C overnight and secondary antibody labeled with horseradish peroxidase at room temperature for 2 h. Finally, samples were counterstained with hematoxylin and visualized with 3,3’-diaminobenzidine (DAB) tetrahydrochloride for 10 min. All the sections were examined and the pictures were taken by digital camera (Leica, ICC50 W) and protein expressions were quantified by Image-Pro Plus 6.0.

For immunofluorescence assays, gingival slides were dewaxed and hydrated, and microwave antigen retrieval was performed in ethylenediaminetetraacetic acid buffer (pH 9.0). Following serum blocking (with 4% goat serum for 40 min), the sections were incubated with the following combinations of primary antibodies: anti‐CD4 (GB13064‐1; Servicebio, Wuhan, China) and anti‐IFN‐γ (ab9657; Abcam, Cambridge, UK), anti‐CD4 and anti‐IL‐4 (ab211212; Abcam), anti‐CD4 and anti‐IL‐10 (ab34843; Abcam), and anti‐CD4 and anti‐IL‐17 (ab79056; Abcam) in a 1:100 dilution overnight at 4°C. After primary antibody incubation, the slides were incubated with the following combination of secondary antibodies: Alexa Fluor 488‐conjugated goat anti‐mouse (ab150117; Abcam) and Alexa Fluor 647‐conjugated goat anti‐rabbit (ab150083; Abcam) for 50 min in the dark. Additionally, a control was performed by omitting the primary antibody. Then, the slides were stained with a 1:200 dilution of 4′‐6‐diamidino‐2‐phenylindole (DAPI) solution (Sigma‐Aldrich, Louis, MO, USA) for 10 min in the dark. Finally, the stained cells were visualized by fluorescence microscopy, and images were collected (NIKON ECLIPSE C1, Nikon DS‐U3, Japan).