Target retrieval solution

Recently, TILs, PD-1, and PD-L1 were investigated in this STS cohort [9 (link)]. We used those results to explore the relationship between VISTA and the PD-1/PD-L1 pathway in STS. TILs between tumour cells were counted per high-power field (HPF) (400× magnification, field of view 0.237 mm²) in H&E-stained TMA slides, as routinely carried out by the pathologist.
As described previously [9 (link)], slides were pre-treated with heat and Target Retrieval solution (S1699, Agilent, Santa Clara, CA, USA) before incubation with the monoclonal primary anti-PD-1 mouse antibody (315M; 1:80; Cell Marque, Rocklin, CA, USA) for 60 min at room temperature. The Vectastain Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA) and the chromogen DAB+ (Agilent) were used for detection and Hematoxylin (Vector Laboratories) for counterstaining.
For PD-L1 staining, slides were pre-treated with heat and the Epitope Retrieval Solution pH8 Novocastra (Leica Biosystems, Wetzlar, Germany) before incubation with the monoclonal primary anti-PD-L1 rabbit antibody (E1L3N; 1:50; Cell Signalling Technology) for 60 min at room temperature.
For CD3 staining, a monoclonal antibody raised in rabbit (SP7; 1:150; Zytomed, Berlin, Germany) was employed according to standard procedures. We used the SignalStain Boost IHC Detection Reagent (Cell Signalling Technology) and the chromogen DAB+ (Agilent) for detection.

Formalin-fixed paraffin-embedded samples were collected at the Institute of Pathology of the LMU Munich^{57 (link)}. We harvested at least two cores per sample with a core-diameter of 1 mm from all blocks to construct tissue microarrays. All EwS samples showed cytogenetic evidence for a translocation of the EWSR1 gene either as determined by fluorescence in situ hybridization and/or qRT-PCR. The samples were reviewed by a reference pathologist. Four-micrometer sections were cut for immunohistochemistry and antigen retrieval was performed with microwave treatment using the antigen retrieval ProTaqs I Antigen-Enhancer (Quartett) for p-MYBL2 or the Target Retrieval Solution (Agilent Technologies) for cleaved caspase 3. In total, 7.5% aqueous H₂O₂ solution (room temperature) and blocking serum from the corresponding kits were used for 20 min for blockage of endogenous peroxidase. Then slides were incubated for 60 min with the primary antibodies anti-p-MYBL2 (1:100 dilution; Abcam, ab76009) and anti-cleaved caspase 3 (1:100 dilution, Cell Signaling, #9661). Afterwards slides were incubated with a secondary anti-rabbit IgG antibody (MP-7401, ImmPress Reagent Kit, Peroxidase-conjugated) followed by subsequent target detection using DAB+chromogen (Agilent Technologies). Slides were counterstained with hematoxylin Gill’s Formula (H-3401; Vector).