Polyclonal rabbit anti s100

S100 protein is expressed by glial cells in the intestine [10] (link) and is used as a glial cell marker. The enteric neural cells express β (III)-tubulin (BTUB) (30). Cultured ISMC and MS were fixed in formalin for 1 hour at room temperature. Samples were incubated with the following primary antibodies: monoclonal mouse anti-α smooth muscle actin (SMA, 1∶50 dilution, Dako, Carpinteria, CA), monoclonal mouse anti-desmin (DES, 1∶50, Dako), monoclonal mouse anti-smooth muscle myosin heavy chain (MHC, 1∶50, Santa Cruz Biotechnology, Dallas, TX), polyclonal rabbit anti-S100 (1∶200, Dako), and monoclonal mouse anti-β (III) tubulin (Abcam, 1∶200, Cambridge, England). After 16 hours of incubation, samples were incubated in a 1∶200 dilution of Alexafluor 488 (green) goat anti-mouse or Alexafluor 594 (red) anti-rabbit IgG (Life Technologies). VectaMount containing DAPI (Vector) was used to visualize the nuclei. All images and videos were taken with an Olympus IX71 microscope with CellSens software (Olympus, Center Valley, PA).

Tissue specimens or cultured cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for IHC analyses. Paraffin-embedded tissue sections from a different cohort (n = 82, WHO grade I = 58, grade II = 21, and grade III = 3) were immunostained using previously described conditions. Briefly, the sections were de-paraffinized using xylene, rehydrated and blocked with 2% goat serum. Samples were subsequently incubated with primary antibody overnight at 4°C and secondary antibody conjugated to Horseradish Peroxidase (HRP) at room temperature for 1 hour. The signals were visualized with an HRP substrate. The following primary antibodies were used: polyclonal rabbit anti-GFAP (glial fibrillary astrocytic protein) (Chemicon International, Temecula, CA), monoclonal mouse anti-EMA (epithelial membrane antigen) (Thermo Scientific, Rockford, IL), polyclonal rabbit anti-S100 and monoclonal mouse anti-vimentin (Dako, Denmark) antibodies. Transglutaminase 2 (TGM2) antibody was purchased from Abcam (Cambridge, UK). At least 4 high-power fields (HPF, 400x) were randomly photographed from the tumor region. In each HPF, individual cell counts were made by single observer and mean percentage were recorded as %/HPF.