Epr1394y

Immunohistochemical staining of TMA sections for PD-L1 protein expression was performed using the FDA-approved rabbit monoclonal antihuman PD-L1 antibody, clone 28-8 (Dako/Agilent; Santa Clara, CA, USA; dilution 1:100). Positivity was defined as ≥1% of tumor cells or macrophages with membranous staining of any intensity for PD-L1, respectively.
TMA sections were further stained with the rabbit monoclonal antibody EPR 1394Y (Abcam, UK; dilution 1:200) and mouse monoclonal antibody C8/144B (Dako/Agilent, USA; dilution 1:200) for MHC I and CD8, respectively. Human tonsil tissue served as staining control on each of the TMA slides. All IHC stainings were performed using a Bond Max automated system (Leica Biosystems; Wetzlar, Germany) in accordance with the manufacturer’s protocol. Evaluation of the data was determined semiquantitatively by three pathologists (KP or SEG and AQ). Discrepant results, which occurred only in a small number of samples, were resolved by consensus review.
For PD-L1 expression on TC and macrophages <1% was defined as negative, whereas ≥1% of expression was categorized as positive.
For CD8 expression <50 lymphocytes/mm² were assessed as negative and ≥50 lymphocytes/mm² were defined as positive considering peritumoral and intratumoral distribution.
For MHC I evaluation <20% were assessed as negative, whereas ≥20% was defined as positive.

Flow cytometry was used to determine the expression of membrane-associated proteins (MHC class I and MHC class II) in 4T1 cell lines treated with F3 (50 μg/ml), dissolved in 0.1% dimethyl sulphoxide, for 24 h. The dose concentration of F3 was previously shown to inhibit 4T1 cells in vitro in a dose- and time-dependent manner [23 (link)]. The primary antibodies used were as follows: Anti-MHCI (Rabbit monoclonal, EPR1394Y; Abcam, Cambridge, UK); Anti-MHCII (Mouse monoclonal, MRC OX-6; Abcam); Rabbit IgG isotype control (EPR25A, Abcam); Mouse IgG1 isotype control (X092701, Dako Denmark A/S). Untreated cells and isotype controls were used as negative controls. Cellular expression of a specific protein was based on the detection of target antigen bound with fluorescent-labeled antibody at equilibrium. For a successful fluorescence activated cell sorting (FACS) of live cells using the flow cytometer, the calibration procedure, reagent preparations and dilution of antibodies were carefully performed to obtain optimum intensity of the fluorescence signal. Data for 10,000 live events were acquired for each sample using a FACS Calibur cytometer for analysis. The FACS results were analyzed using the FlowJo v.X.0.7 software (Tree Star Inc., Ashland, OR, USA).