Alk 5a4

Subsequently, these samples were analyzed for ALK expression. FFPE sections (5-μm thickness) were stained using ALK 5A4 (Leica) with EnVison Flex+, mouse high pH detection reagents on an Autostainer Lin 48 instrument (Dako, Glostrup, Denmark). The sections were subsequently incubated in high pH buffer (20 min, 97°C; PT-Link, Dako), peroxidase blocking buffer (5min), primary antibody (1:50, 30 min), mouse-enhanced polymer-based linker (30 min), mouse secondary antibody (20 min), diaminobenzidene (5 min) and haematoxylin (5 min) as previously described [40 (link)]. ALK expression was assessed independently by one pathologist (PP) and one scientist (KZ). IHC ALK positive samples were evaluated with Fluorescence in-situ hybridization (FISH). High ALK- expressors were analyzed with next generation sequencing to detect ALK mutations.

Immunohistochemical staining was performed in the Johns Hopkins Immunopathology Laboratory on formalin-fixed, paraffin-embedded tissue sections, using Ventana Benchmark automation and the Ultra View detection kit (Ventana Medical Systems, Tucson, AZ, USA) as previously described [26 (link),27 (link)]. All immunostains were performed at the time of diagnosis for primary or recurrent tumor. Antibodies from Cellmarque, Hot Springs, AZ, included: c-Kit (YR145), DOG-1 (SP31), CAM5.2 (B22.1&B23), Calretinin (Polyclonal), Cyclin D1 (SP4); antibodies from Ventana, Tucson, AZ, USA, included: CD34 (QBE/10), CD68 (KP-1), SMA (1A4), AE1/AE3 (pck-26), Actin (MOUSE), S100 (4C4.9), ER (SP-1), Melan A (A103); antibodies from DAKO, Carpinteria, CA, USA, included Desmin (D33) and Caldesmon (h-CD); antibodies from Abcam, Cambridge, MA, included ALK (5A4), Cathepsin K (3F9), and SDHB (21A11AE7); antibodies from Leica, Bannock Burn, IL, USA, included CD10 (MS/56C6) and PR (1E2); the rest of the antibodies included SOX-10 (N-20, Biocare, Concord, CA, USA), HMB-45 (HMB45, Novocastra, Newcastle upon Tyne, UK), Inhibin (R1, Serotec, Raleigh, NC), STAT6 (D-1, Santa Cruz, Santa Cruz, CA, USA), and SF-1 (n1665, Invitrogen, Waltham, MA, USA).