Fitc anti human hla a2

Histological melanoma tumor sections were stained with hematoxylin and eosin stains (H&E) or antibodies for immunofluorescence (IF) and immunohistochemistry (IHC). Primary anti-human cleaved caspase 3 (Cell Signaling Technology) monoclonal antibody was used for IHC. Alex Fluor™ 647 anti-human NY-ESO-1 (BioLegend, CA, USA) and FITC anti-human HLA-A2 (BioLegend, CA, USA) were used for IF. The sections were imaged with a TissureFAXS PLUS, and images were analyzed with TissueFAXSViewer.

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.