Clone d33

A complete necropsy was performed on twelve subjects and biopsy samples taken from the other four degus. Tissue samples were fixed in 10% neutral buffered formalin and routinely processed for paraffin embedding. Four-micrometers thick sections were stained with Hematoxylin and Eosin, Goldner’s Trichrome and Periodic-Acid Schiff. Immunohistochemistry was performed on the selected sections using mouse monoclonal antibodies anti-human vimentin (1:50; clone V9, Dako, Glostrup, Danmark), anti-human cytokeratin AE1/AE3 (1:100; Dako, Glostrup, Danmark), desmin (1:50; clone D33, Dako, Glostrup, Danmark) and anti-human c-kit (CD117) (1:300; Dako, Glostrup, Danmark). Antibody binding was detected by EnVision Detection System Peroxidase/DAB +, Rabbit/Mouse (DAKO, Glostrup, Danmark). Tumours were classified according to the WHO classifications of tumors of domestic animals [15 ,16 ,17 ,18 ].

The myogenic purity of the cultures was monitored by determining the expression of desmin, a cytoskeletal protein that is expressed only in myogenic cells and not in fibroblasts. The number of desmin-positive cells, represented as a percentage of the total number of nuclei, was determined as the myogenic purity of the cell culture, and at least 500 cells were counted. Immunocytochemistry was performed using an antibody specific for desmin, at a dilution of 1:50 (clone D33; DAKO, Denmark). The cells were washed with × 1 phosphate-buffered saline (PBS) and fixed with 100% ethanol for 10 min. The fixation agent was removed by washing three times with × 1 PBS for 5 min. Non-specific binding sites were blocked with 1% FBS diluted in PBS for 30 min. The cells were then incubated with primary antibody against desmin. Specific antibody binding was detected using Alexa Fluor 488 (Invitrogen, USA) directly coupled to the secondary antibody at a dilution of 1:500. The nuclei were fluorescently detected by Hoechst staining (Sigma, USA) at a dilution of 0.0001% w/v. All images were digitalised using ImageJ software.

Formalin-fixed tumors were embedded in paraffin using standard procedures. The 5 μm sections were processed and stained with antibodies directed against Ki67 (1:100; Dako, clone MIB-1), desmin (1:80; Dako, clone D33) and von Willebrand Factor VIII (1:100; Biocare Medical, #CP039B).

The central pathology review was performed conjointly by 3 neuropathologists (ATE, PV, EL), a pathologist expert in soft tissue tumors (FL), and a pathologist expert in vascular lesions (MW). For the CNS cases, additional immunohistochemical stainings were performed on paraffin-embedded sections including: CD34 (1:40, clone QBEnd10, Dako, Glostrup, Denmark), smooth muscle actin (SMA) (1:1500, clone 1A4, Dako, Glostrup, Denmark), Desmin (1:400, clone D33, Dako, Glostrup, Denmark), h-caldesmon (1:100, clone h-CALD, Santa Cruz Biotechnology, Dallas, USA), PS100 (1:6000, polyclonal, Dako, Glostrup, Denmark), GFAP (1:200, clone 6F2, Dako, Glostrup, Denmark), neurofilament (1:25, clone 2F11, Dako, Glostrup, Denmark), STAT6 (1:200, clone YE361, Abcam, Cambridge, UK), SSTR2a (1:200, clone UMB1, Abcam, Cambridge, UK) and EMA (1:200, clone GM008, Dako, Glostrup, Denmark). All immunohistochemical stainings were performed in an automatic closed immunostainer (Omnis automate). Orcein staining was also performed.