Benchmark xt automate

U251-MG and U87-MG cell lines were grown as a monolayer on glass coverslips in 24-well plates in DMEM with 10% FCS. For GBM CSCs, immunofluorescence experiments were performed on 8-day-old spheres or on cells cultured on poly-DL-ornithin as explained above. Primary antibodies A2B5 (mouse IgM, clone 105, 1/1000, kindly provided by G. Rougon, Marseille, France), anti-ST8SIA3 (rabbit IgG, 1/1000, Sigma-Aldrich), and anti-Ki67 (mouse IgG, clone Mib1, Dako, Les Ulis, France) were incubated for 1 h at room temperature. Fluorochrome-conjugated secondary antibodies Alexa fluor 568 anti-mouse IgM, Alexa fluor 488 anti-rabbit IgG, Alexa fluor 568 anti-mouse IgG, or Alexa fluor 568 anti-goat IgG (Molecular Probes, Eugene, OR, USA) were incubated at 2 µg/mL for 1 h at room temperature together with Hoechst 33342. All the images were obtained using a Zeiss AXIO-Observer Z1 microscope (Carl Zeiss SAS, Marly-le-Roi, France). Conventional immunohistochemistry for cleaved caspase-3 (C92-605 clone, 1/500, BD Biosciences, Franklin Lake, NJ, USA ) and Ki67 (30-9 clone, Ventana Medical Systems, Illkirch, France) was performed using the Benchmark XT automate (Ventana Medical Systems) according to the manufacturer’s instructions. Sections without primary antibody served as a negative control.

For each subcutaneous tumor, 4 μm thick paraffin sections were prepared and stained with hematoxylin and eosin for histological study. All brains were sectioned and stained with hematoxylin and eosin to attest to the presence of a tumor. Conventional immunohistochemistries for Ki-67 (30–9, Ventana Medical Systems, Illkirch, France) and GFAP (EP672Y, Ventana Medical Systems) were performed using the Benchmark XT automate (Ventana Medical Systems) according to the manufacturer's instructions.

Tumors were excised, weighed and sectioned. For each subcutaneous tumor, 4 μm thick paraffin sections were prepared and stained with Ki-67 (Ventana Medical Systems, France). Staining was performed using the Benchmark XT automate (Ventana Medical Systems) according to the manufacturer’s instructions.