Anti pd l1 66248 1 ig

Patient tissue samples and mouse tumor tissues were fixed in neutral buffered formalin (10%), paraffin-embedded, and sectioned at 4 μm thickness. For IHC, the samples were incubated with the following primary antibodies at 4 °C overnight: anti-USP8 (27791-1-AP, Proteintech), anti-PD-L1 (66248-1-Ig, Proteintech), anti-CD8a (98941, Cell Signaling Technology), anti-Granzyme B (44153 S, Cell Signaling Technology), anti-cleaved caspase-3 (9661, Cell Signaling Technology), and anti-Ki67 (12202, Cell Signaling Technology). The next day, the sections were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP). The samples were visualized using a diaminobenzidine (DAB) chromogen kit (BDB2004, Biocare, Pacheco, CA, USA). Image Scope software (Leica Biosystems, Wetzlar, Germany) was used to capture representative images of the tumors. The quantitative results for IHC staining were obtained using ImageJ software and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Microarray slides of PDAC tissue were immunostained using antibodies against USP8 (27791-1-AP, Proteintech) and CD274 (ab205921, Abcam). 3DHISTECH QuantCenter 2.1 software (3Dhistech, Budapest, Hungary) was used to quantify the IHC results.

Tumor cell digestion was performed as previously described for the isolation of primary RCC cells. After filtering, tumor cells were resuspended in PBS containing 5 × 10^‐3m EDTA and 1% FBS. For measurement of PD‐L1 expression, non‐immune cells were removed via density gradient centrifugation, and the remaining cells were stained with anti‐PD‐L1 (66248‐1‐Ig, Proteintech) or isotype‐control antibodies. After three washes with PBS, cells were stained with Alexa Fluor 488‐conjugated goat anti‐mouse IgG (A‐11001, Invitrogen) for 20 min. For detection of cytotoxic cytokines production, cells were treated with 50 ng mL^‐1 phorbol 12‐myristate 13‐acetate (PMA), 1 × 10^‐6m ionomycin and protein transport inhibitor (BD) for 6 h at 37 °C. Cells were then fixed and permeabilized with a Fixation and Permeabilization Solution Kit (BD, 554714) following the manufacturer's instructions, and cells were then stained with the indicated primary antibodies. For measurement of HLA‐A2 expression, tumor cells were stained with FITC anti‐human HLA‐A2 (343303, Biolegend) or isotype‐control antibodies. For apoptosis analysis, cells were collected and evaluated by the Annexin V‐APC/PI apoptosis kit (AP‐107, Multi Science) according to the manufacturer's instructions.
Samples were analyzed with a Beckman CytoFLEX Flow cytometer (Beckman Coulter, USA), and FlowJo10 software was used to analyze the data.