Ab39253

The GBM cell lines, U87, A172, and U251, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were regularly verified for cell morphology and growth characteristics by microscopic analysis. All the cells were cultured in 10% FBS-supplemented DMEM and maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The procedures to obtain and stain glioma stem/progenitor (GSP) cells from U87 cells were as previously described [15 (link)]. The growth factor EGF and bEGF were purchased from PeproTech; B27 was purchased from Gibco. Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay. Slides were stained with DAPI (Beyotime Biotechnology) for 5 min prior to examination using a confocal microscope.

With the approval of the institutional ethics committee, forty-eight patients with primary GBM whom underwent tumor resection in the Neurosurgical Department of Shanghai Tenth People’s Hospital from Jan 2011 to Dec 2013 were given informed consent and enrolled in this study. The median age was 58 years (range, 40–75); 20 patients were male and 28 female. All patients received surgical treatment and specimens were fixed with 10% formalin, embedded in paraffin, and examined histopathologically. Immunohistochemistry was done as previously described [13 (link)], and the specimens were not affected by the chemotherapy. Primary antibodies were used as follow: rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam). The percentage of tumor cells with Gli1 nuclear staining to total cancer cells being > 10%, it was judged to be Gli1-positive. With the ratio of tumor cells stained with MGMT > 5%, it was judged to be MGMT-positive [14 (link)]. Sections from the same tissues without application of the primary antibodies were used as negative controls.