Cox 2 polyclonal antibody

To identify the origin of primary FM cells obtained from muscular tissue and FISS‐derived cells, ICCs were incubated with vimentin, desmin and anti‐alpha‐smooth muscle actin (α‐SMA) antibodies. The cells were seeded onto a 96‐well plate (4.5 × 10³ cells/well) and incubated at 37°C for 24 h to reach 80%–90% confluence. After removal of the culture medium, the cells in each well were fixed with 80% acetone at −20°C for 10 min, air‐dried at RT and stored at −20°C for subsequent use. One hundred microliters of anti‐vimentin antibody (1:800 dilution) (Dako), anti‐desmin antibody (1:200 dilution) (Dako) and α‐SMA antibody (1:800 dilution) (Dako) were added to each well and incubated for 1 h at RT. After washing with PBS, the EnVision® + Dual Link System‐HRP (DAB+) (Dako) was used following the manufacturer's protocol. The images were evaluated using an inverted microscope (Eclipse TS 100; Nikon) by two pathologists. To quantify the positive cells in the tumour, five high‐power fields were randomly selected and captured and these pictures were analysed using ImageJ software (NIH). The ratio was calculated by dividing the positive cell count by the total cell count. To detect the expression of COX‐2 in FISS and FM cells, ICC was conducted as previously described. The primary antibody was replaced with a COX‐2 polyclonal antibody (Cayman) diluted at 1:200 in PBS.