Normal mouse serum

Glass slides containing 12 μm coronal sections were air dried for 30 min. They were then immersed in PBS (Fresenius Kabi)‐formalin (Sigma‐Aldrich) 10% v/v solution, for 10 min, washed three times with PBS and blocked with PBS‐NaN₃ (Merck) 0.1% w/v‐H₂O₂ (Supelco) 0.02% v/v for 10 min and washed again twice with PBS. After this, they were immersed in PBST (PBS‐Tween 20 [Merck] 0.1% v/v), and blocked in PBST‐BSA (Sigma‐Aldrich) 5% w/v for 30 min. Slides were incubated with antibodies solution (Anti‐NeuN [ab104225] 1:500, Abcam; RRID:AB_10711153) overnight at 4°C. After washing three times with PBST, they were incubated in secondary antibody mix (Rabbit EnVision; Dako) +5% normal mouse serum (Dako) for 30 min at room temperature, and then washed three times with PBST. Sections were then stained with liquid DAB+ substrate chromogen system (Dako) for 1 min and washed with deionized water, and counterstained with Mayer's hemalum (Sigma‐Aldrich) solution 1:4 for 45 s. Finally, they were dried in hot air and slip covered. Digital images from Corpus Callosum were obtained as described in Amaya et al.²³ Images were processed with FIJI software (http://fiji.sc, RRID:SCR_002285). From each image, blue channel was used to display immunopositive signal. Cingulate cingulum was parcellated in each image, and immunopositive cell bodies within were counted manually.

The tissue microarray was purchased from US Biomax and immunohistochemically stained as described previously (Tanaka et al. 2011 (link)). Briefly, a tissue array consisting of cores from formalin-fixed paraffin-embedded tumors and normal brain blocks was deparaffinized by immersion in 0.01 M citrate buffer (pH 6) for 30 min in a pressure cooker to promote antigen retrieval. Peroxidase activity was quenched with 3% hydrogen peroxide in water, and primary antibodies were applied for 16 h at 4°C followed by biotinylated secondary antibodies and the avidin–biotin complex. Primary antibodies against survivin and phospho-p65 (Cell Signaling Technologies) were used. Negative control slides received normal mouse serum (Dako) for the primary antibody step. Slides were counterstained with Harris hematoxylin. Staining intensity was scored independently by two neuropathologists based on a scale of three categories indicated as low for negatively stained specimens, medium for weakly positive samples, and high for strongly positive spots. Statistical significance between normal and tumor staining was calculated using the independent t-test. Statistical analyses were performed with the two-tailed Student's t-test to determine the statistical significance between independent samples, with P-values of <0.05 considered statistically significant.