Ab255598

For immunohistochemistry (IHC), HCC patient tissue microarrays (TMA) were produced by Zhuoli Biotech (Shanghai, China) and were stained with the indicated antibodies. Multi-color IHC assays were performed using the respective kits (Panovue, Beijing, China). Immunofluorescence images were acquired using a confocal microscope (Leica LAS AF Lite 2.6.0). Signal intensities were quantified by ImageJ 1.8.0. Antibodies for IHC and multi-color IHC were: BLIMP1 (ab198287, Abcam, 1:500), USP22 (ab195289, Abcam, 1:1000), SPI1 (ab227835, Abcam, 1:1000), PD-L1 (ab205921, Abcam, 1:1000), CD8 alpha (ab245118, Abcam, 1:1000), and GZMB (ab255598, Abcam, 1:3000). Antibodies used for immunofluorescence were USP22 (ab235923, Abcam, 1:200), SPI1 (ab88082, Abcam, 1:200), CD8 alpha (#GB11068, Servicebio, 1:200), and PD-L1 (ab213480, Abcam, 1:200).

Tumor tissue was also stained against granzyme B after deparaffinization and rehydration. Antigen retrieval was carried out using standard heat-based antigen retrieval techniques [18 (link)]. Immunofluorescence staining was carried out according to previously published procedures [15 (link)]. After blocking with 5% goat serum, rabbit anti-mouse granzyme B primary antibody (ab255598; Abcam, Cambridge, UK) was incubated overnight at 4 °C. On the following day, washing was carried out followed by incubation with AlexaFluor 647 conjugated goat anti-rabbit IgG secondary antibody (A32733; ThermoFisher, Waltham, MA, USA) at room temperature in a moist dark chamber for 1 h. DAPI (4′, 6-diamidino-2-phenylindole) was used for staining cell nuclei. Semi-quantitative data of immunofluorescence were retrieved by measuring total fluorescence in the red channel (granzyme B) divided by area (integrated density). All images were acquired using Biotek Cytation 5 Cell Imaging Multi-Mode Reader and analyzed through Biotek Gen5 software (Agilent, v3.11, Santa Clara, CA, USA).

Paraffin-embedded lung sections were sectioned and processed for standard H&E's or immunohistochemistry (IHC) using antibodies to CD99 (Abcam #ab27271) followed by ImmunoHistoMount (ImmunoBioscience #AR-6503-01) staining. Immunofluorescence was performed using primary mouse anti-CD99 antibody [HO36-1.1] (Abcam #ab212605) and anti-Granzyme B antibody [EPR22645-206] (Abcam #ab255598). Secondary anti-mouse Alexa 594 (Thermo Scientific #A11005) and anti-rabbit Alexa 488 (Thermo Scientific #A11008) antibodies were used for detection. Images were acquired using Zeiss LSM 800 Airyscan system controlled with Zen Blue software (version 2.6).

Cells and exosomes were treated with lysis buffer (1% Triton X-100, 0.1% SDS, 0.1 M Tris HCl, pH 7, 1 mM PMSF, Sigma-Aldrich). Samples were then subjected to protein quantification using the BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples (20 μg/well) were loaded onto 10% SDS-PAGE gels and separated. Following transfer, the membranes were blocked with 5% skim milk or BSA for 1 h and washed twice with PBST, incubated overnight at 4 °C with the corresponding primary antibody, and then washed three times with PBST. The membranes were incubated with the corresponding horseradish peroxidase-coupled secondary antibodies (Cell Signaling Technology) for 2 h at room temperature. The immunoreactive bands were detected on a Mini-Medical/90 Developer (Image Works) using ECL chemiluminescence substrate (GE Healthcare). The following primary antibodies were applied: Calnexin (ab133615, Abcam), CD9 (ab263019, Abcam), CD63 (ab134045, Abcam), Hsp70 (ab181606, Abcam), TSG101 (ab125011, Abcam), GAPDH (390035, ZENBIO), Granzyme B (ab255598, Abcam), and Perforin (ab256453, Abcam).

The antibodies used in this study were as follows: anti-cleaved caspase-3 (#9661, Cell Signaling Technology), anti-mouse CD8a (ab217344, Abcam), anti-Foxp3 (ab215206, Abcam), and anti-mouse granzyme B (ab255598, Abcam). Tissue slides were deparaffinized, incubated with 3% H₂O₂ in water for 10 min to quench endogenous peroxidase activity, and subjected to heat mediated antigen retrieval with Antigen Unmasking Solutions (H3300, Vector Laboratories, Burlingame, CA). Tissue sections (3-µm thickness) were stained with the HRP-conjugated avidin-biotin complex (ABC) from the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) and DAB chromogen (Vector Laboratories) and counterstained with hematoxylin.
Staining for immune cells was positive when detected in the tumor-infiltrating lymphocytes (TILs) and was evaluated using a microscope (OLYMPUS BX53, Tokyo, Japan). Regarding the detection of TILs, the tissue was viewed at 40× magnification, and the area with the highest density of CD8⁺, GzmB⁺, and Foxp3⁺ TILs within the malignant cells was counted at 400× magnification (no. of TILs/high-power field). The average number of tumor-infiltrating immune cells in five high-power fields was included in the evaluation [30 (link)].

Tumor tissues were fixed in 4% paraformaldehyde for 24 h and paraffin embedded. Longitudinal cross sections of the mouse tumor tissue from these blocks were obtained and deparaffinization was performed by warming the slides at 65 °C for 30 min. The slides were then immersed in xylenes and next immersed in 100%, 95% then 70% ethanol for 15 min each. The slides were washed under running tap water and then incubated in a 1% hydrogen peroxide/methanol solution for 10 min. The slides were rinsed with distilled water and rinsed in PBST (Phosphate-Buffered Saline containing 0.05% Tween-20), then incubated with an anti-mouse-PD-L1 (Abcam #ab213480), anti-mouse IRF1 (CST #8478), anti-mouse CD8 (Abcam #ab217344), anti-mouse IFN-γ (Abcam #ab216642), anti-mouse-Cl. Caspase 3 (Proteintech #19677-1-AP), anti-mouse-PCNA (CST #2586), anti-mouse GZMB (Abcam #ab255598) at room temperature for 1 h. The slides were rinsed with PBST and incubated with HRP-labeled Polymer AntiRabbit (CST) at room temperature for 30 min. After a rinse with PBST, the slides were incubated with TSA (Tyramide signal amplification) for visualization. Next, the slides were washed with distilled water and then dehydration with 75%, 95% and 100% alcohol. Last, put the slides in xylene solution for 20 min and treated with neutral gum.

The antibodies used in this study were as follows: anti-CD11c (ab52632, Abcam) and anti-mouse granzyme B (ab255598, Abcam). The tissue slides were deparaffinized and subjected to heat-mediated antigen retrieval with Antigen Unmasking Solutions (H3300, Vector Laboratories, Burlingame, CA). Incubate tissue slides with 5% horse serum for 10 min. Tissue Sect. (3 μm thick) were stained with indicated primary antibodies and the PE-conjugated secondary antibodies, and counterstained with DAPI [27 (link), 28 (link)].
Staining for immune cells was positive when detected in the tumor-infiltrating lymphocytes (TILs) and was evaluated using a microscope (OLYMPUS BX53, Tokyo, Japan). For the detection of TILs, the tissue was viewed at 40× magnification, and the area with the highest density of CD11C⁺ and GzmB⁺ TILs within the malignant cells was counted at 400× magnification (no. of TILs/high-power field). The average number of tumor-infiltrating immune cells in five high-power fields was included in the evaluation [29 (link)–31 ].