Sox10 clone bc34

Tissue sections (5 μm thickness) were obtained from archival FFPE tumor blocks from patients treated for melanoma at Vanderbilt University Medical Center. All participants provided informed consent for use of tissue specimens and the study was approved by the Vanderbilt Institutional Review Board (IRB Approval numbers 100178 and 030220). Participants all received immune checkpoint inhibitors as second or third line therapy. Individual demographic information, therapies and responses, biopsy site(s) sampled and other relevant information are provided in supplemental Table S1. Samples were fixed in 10% buffered neutral formalin, processed and paraffin-embedded by standard methods in automated tissue processor. Five-micrometer sections were subjected to hematoxylin and eosin (H&E) staining and dual IHC using antibodies against PD-L1 (clone E1L3N, Cell Signaling Technology, Danvers, MA) and SOX10 (clone BC34, Biocare Medical, Pacheco, CA). Sections analyzed by hematoxylin and eosin (H&E) staining and IHC were serially adjacent to those analyzed by MS.

For a representative view of the tumors, an average of 3 cores of 1 mm per tumor were mounted on tissue microarrays. For each tissue block, a 4 μm section was cut and dried at 60°C for 1 hour. The sections were deparaffinized and pretreated in the PT-Link (Dako) with Target Retrieval Solution buffer at pH 9. The following steps (except for the primary antibody staining) were performed in the Dako staining equipment (Autostainer plus) with Dako kit K8010 solutions: peroxidase block (5 minutes), EnVision HRP-conjugated polymers for 30 minutes, DAB substrate-chromogen solution twice for 5 minutes, and counterstaining with hematoxylin for 4 minutes. Between the steps, the sections were rinsed with washing buffer. Finally, the sections were dehydrated and mounted with Pertex mounting medium (Histolab). The primary antibodies used from Dako were MITF (clone C5, Biocare) in 1:100 dilution and SOX10 (clone BC34, Biocare) in 1:100 dilution. Cores were scanned and uploaded in PathXL (Philips) platform to be further analyzed.
Mouse brains from in vivo assays were processed in the same way and stained by anti-mitochondria antibody 113-1 (catalog ab92824, Abcam) to visualize human melanoma metastatic cells.

Excised tumour spheroids were fixed in buffered formalin (Histofix). Formalin-fixed paraffin-embedded sections were cut at a thickness of 3 µm. The Melan A (clone A103, Roche, Ventana) and SOX10 (clone BC34, Biocare Medical) immunohistochemistry analysis was performed using the Ventana BenchMark ULTRA system (Ventana Medical Systems, Tucson, AZ, USA). The immunohistochemistry (IHC) analysis for Melan A was performed using the following protocol: pretreatment CC1 was performed for 36 min at 95 °C, and incubation occurred for 24 min at 27 °C. For SOX10, CC2 pretreatment was performed for 40 min at 90 °C, and incubation occurred for 32 min at 36 °C. Visualisation was conducted using the OptiView DAB System (Ventana Medical System). Thereafter, slides were scanned using a Leica DM4000B microscope with a Leica DFC290 camera and analysed using the Leica Suite version 2.8.1 (Leica Microsystems, Wetzlar, Germany). The slides were reviewed by a board-certified pathologist.