Ab134114

The total protein from tissues from extracted using Phenyl methylsulfonyl Fluoride (PMSF)-containing protease inhibitor (p0100, Solarbio, Beijing, China) in strict accordance with the instructions. The supernatant was extracted, and the protein concentration of each sample was determined by bicinchoninic acid (BCA) kit (23,227, Thermo Fisher). The protein was quantified according to different concentrations, transferred to polyvinylidene fluoride (PVDF) membrane by polyacrylamide gel electrophoresis, and sealed with 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane underwent incubation with primary antibodies against CD19 (ab134114; 1: 2000, Abcam, Cambridge, United Kingdom) and β-actin (ab8226; 1: 5,000, Abcam) overnight at 4 °C, and then secondary antibody horseradish peroxidase (HRP) labeled goat anti rabbit Immunoglobulin G (IgG; ab205718; 1: 20,000, Abcam) at room temperature for 1.5 h, followed by color development using developer (NCI4106, pierce, Rockford, IL, Unites States). ImageJ 1.48u software (Bio-rad, Hercules, CA, Unites States) was used for protein quantitative analysis, and the relative protein content was expressed by the gray value of the corresponding protein band and the gray value of β-actin. Each experiment was conducted in triplicates.

The myometrium samples were embedded in paraffin and sliced into 5 μm thickness. To perform immunohistochemical staining, the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (ab64264, Abcam) was used, following the manufacturer's instructions. Peroxidase quenching was used for the hydrogen peroxidase block. Tissue sections were incubated overnight in a moist chamber at 4°C with primary antibodies CD68 (1:500, ab201340, Abcam), CD3 (1:150, ab135372, Abcam), CD56 (1:20000, ab75813, Abcam), CD19 (1:100, ab134114, Abcam) or CD66 (1:100, ab197678, Abcam). After primary antibody incubation, the tissue sections were incubated with Biotinylated Goat Anti‐Polyvalent secondary antibody for 1 hour. The diaminobenzidine tetrahydrochloride (DAB) chromogen was used for the production of brown colouration. Images were captured by a Leica DMi8 fluorescence microscopy.

Immunohistochemistry of patient tumor tissue was performed on 5 μm paraffin sections using antibody against HLA‐A (abcam, ab52922, 1:200), HLA‐B (abcam, ab225636, 1:200), HLA‐C (abcam, ab193432, 1:200), and CXCR4 (abcam, ab181020, 1:200). Immunohistochemistry of zebrafish model tissues was performed on 3 μm agarose sections,²³ stained with H&E or primary antibodies of anti‐human CD19 (abcam, ab134114, 1:200) and anti‐human CXCR4 (abcam, ab1670, 1:150). Protein expression levels were recorded according to percentage of stained cells. DLBCL exhibiting a >50% reduction of HLA expression (evidently reduced relative to surrounding non‐malignant cells) was categorized as reduced HLA expression, otherwise it was classified as positive expression, as previously reported.²⁴ CXCR4 staining ≥20% was referred as positive.
For immunofluorescence, zebrafish model tissues were fixed in 4% formaldehyde and stored in methanol at −20°C. Tumor cells were labeled by Dil, and nuclei were counterstained with DAPI. Pictures were captured in a Zeiss880 microscope.

Multiplex immunofluorescence staining was performed by the Opal 4-Color IHC kit (Abs50028-20T, Absin). The liver sections were processed by antigen retrieval. The primary antibody of TSPAN1 was incubated at 37°C for 1 hour. Then, the sections were washed and incubated with HRP-conjugated secondary antibody for 20 minutes at room temperature, what followed was adding TSA dye 520. The second antibody CD19 (ab134114, Abcam) was incubated overnight at 4°C, what followed was adding TSA dye 650. The last antibody CXCR3 (ab288437, Abcam) and TSA dye 570 were added sequentially. The nuclei were stained with DAPI.

Paraffin sections were dewaxed and hydrated, and antigen repair was performed with 0.01 mol/L citrate buffer. Paraffin sections were blocked with endogenous peroxidase blockers. Nonspecific antigen was blocked with 10% calf serum. Subsequently, paraffin sections were incubated overnight at 4°C with the rabbit antibodies against human CD4 (Abcam, ab133616, Cambridge, U.K., 1:250), human CD8 (Abcam, ab4055, Cambridge, U.K., 1:200), human CD19 (Abcam, ab134114, Cambridge, U.K., 1:20), mouse CD14 (Abcam, ab182032, Cambridge, U.K., 1:1000) and mouse CD56 (Abcam, ab220360, Cambridge, U.K., 1:2000). The following day, HRP-labeled goat anti-rabbit secondary antibodies (Abcam Cambridge, U.K.) were incubated with the tissues at room temperature for 1 h. After staining with DAB chromogenic solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China) at room temperature, the sections were observed under a light microscope. All experiments were repeated three times.