Anti vimentin antibody

Tissue sections were stained with an anti-ATRX polyclonal antibody (1: 300 dilution, ATLAS Antibodies AB, Bromma, Sweden), anti-GFAP (6F2) monoclonal antibody (1: 200 dilution, DAKO, Glostrup, Denmark), anti-IDH1 R132H (H09) monoclonal antibody (1:300 dilution, Dianova, Hamburg, Germany), anti-Ki67 (MIB-1) antibody (1:100 dilution, DAKO), anti-H3K27 M polyclonal antibody (1:700 Millipore, Temecula, USA), anti-p53 monoclonal antibody, DO-7 code M7001 (1:100 dilution, DAKO), anti-PHH3 antibody (1:100 dilution, Cell Marque, Rocklin, CA, USA) and anti-vimentin antibody (1:500, DAKO) (Supplementary Table 2). IHC staining was performed using a standard avidin–biotin-peroxidase method with a BenchMark ULTRA system (Roche Diagnostics, Indianapolis, US). The positive control was known positive tissue, and entrapped positive cells were used. For the negative control, the primary antibody was omitted.
ATRX gene mutations were scattered throughout exons and introns, making some sites difficult to capture using NGS. Since the initial version of our customized brain tumor panel could not detect full variants of ATRX mutations, we relied on ATRX IHC, but upgrade version of BTP panel could detect all the mutations.

At the end of the study, animals were sacrificed by sodium pentobarbital overdose (180 mg·kg⁻¹) and their hearts were excised and snap frozen in isopentane cooled with liquid nitrogen. Contiguous 8 μm sections were obtained with a cryostat at − 22 °C for autohistoradiography and histological staining, respectively.
Distribution of ¹⁸F-FDG activity was recorded with an autohistoradiography system dedicated to the detection of electrons and positrons (µImager™, Biospace, France).22 (link) For Hematoxylin-Eosin-Safran (HES) staining, the sections were fixed in 95% ethanol, stained with hematoxylin for 1 minute, eosin and safran for 30 seconds each, before being dehydrated in ethanol 100% and xylene. For the Masson trichrome staining, the sections were fixed by immersion in Bouin solution for 15 minutes and picric acid for 5 minutes. The nuclei were stained with Weigert hematoxylin for 10 minutes, cytoplasm and smooth muscle with Biebrich solution and collagen fibers by immersion for 5 minutes in aniline blue.
For further immunohistology analyses, adjacent 5 µm sections were fixed with paraformaldehyde 4% (VWR, Fontenay-sous-Bois, France), incubated with a rabbit polyclonal antibody to determine macrophage infiltrates (anti-Vimentin antibody; 1:500; Dako, Les Ulis, France).

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.