Ab288724

Tissues were embedded in the optimal cutting temperature (OCT) compound and sectioned at 100 µm or 20 µm with a cryostat. Frozen sections were permeabilized and blocked in PBS containing 5% normal goat serum (SL038, Solarbio) and 0.3% Triton X-100 (V900502, Vetec) for 1 h at room temperature. The sections were then incubated with primary antibody against CD4 (1:50; ab288724, Abcam), CD8 (1:100; ab237709, Abcam), B220 (1:100; 14-0452-86, eBioscience), Laminine (1:100; MA106100, Invitrogen), P-Glycoprotein (1:72; MA1-26528, Invitrogen), GFAP (1:400; 3670S, CST), Il-1β (1:200; P420B, Invitrogen), or Fibrinogen (1:100; NBP2-80414, Novus) overnight at 4 °C. After 3 washes in PBS, sections were incubated with Alexa Fluor^® 488-conjugated anti-rabbit IgG (H + L) (1:500; 4412S, CST) and Alexa Fluor^® 555-conjugated anti-rat IgG (H + L) (1:500; 4417S, CST), or with Alexa Fluor^® 594-conjugated anti-mouse IgG (H + L) (1:500; 8890S, CST), for 1 h at room temperature. In addition, DAPI (1:2000; Sigma, 32670) was used to stain cell nuclei. Slides were mounted with Fluoro-Gel (17985-10, Electron Microscopy Sciences, Hatfield, PA, USA) and imaged using an Olympus IX71 fluorescence microscope (Olympus).

The tumor, liver, and colon were collected from the treated mice, fixed with 4% paraformaldehyde, and embedded. The tissue sections were prepared and subjected to H&E staining. For immunohistochemistry staining, the tumor sections were antigen retrieved using citrate buffer (62 (link)) and stained with Ki-67 (1:200; Servicebio, Nanjing, China) and secondary antibody (1:200; Servicebio). The sections were scanned and analyzed using Caseviewer (3DHISTECH Ltd., Budapest, Hungary).
For the quantification of the infiltration of helper T cells (CD3⁺ CD4⁺), macrophages (CD163⁺ CD68⁺), cytotoxic T cells (CD3⁺ CD8⁺), and Treg cells (CD4⁺ FOXP3⁺), the tumor sections were labeled with IF staining. The antibodies used in the study were follows: CD163 (16646-1-AP; Proteintech, Wuhan, China), CD68 (66231-2-Ig; Proteintech), CD3 (17617-1-AP; Proteintech), CD4 (ab288724; Abcam, UK), CD8 (66868-1-Ig; Proteintech), and FOXP3 (22228-1-AP; Proteintech). After dewaxing, the tissue sections were treated with the citrate antigen retrieval solution (Beyotime) and incubated with 5% bovine serum albumin solution for 30 min. The primary antibodies were added to tissue slices at 4°C overnight, followed by secondary antibodies incubated for 1 hour at room temperature. The sections were scanned and analyzed using Pannoramic MIDI (3DHISTECH Ltd.).

Paraffin-embedded section (3 µm thick) were dewaxed in xylene and heated in 0.01 M sodium citrate buffer (pH = 6.0) in a microwave at 60 °C for 20 min for antigen retrieval. Tissue sections were then blocked in 0.3% H₂O₂ for 10 min to remove endogenous peroxidase activity, followed by incubation with primary antibody against CD4 (1:1000; ab288724, abcam, Cambridge, MA, USA), CD20 (1:50; ab78237, abcam), or CD138 (1:100; ab130405, abcam). The target was visualized with a DAB staining kit (GK600710, Gene Tech, Shanghai, China).