Rpn202

Histological analysis and immunohistochemical staining were performed as described previously 15 (link), 16 (link). Briefly, after deparaffinization, rehydration and antigen retrieval, tissue slides (4μm thick) were blocked with 3% H₂O₂ for 10 min and incubated with primary antibodies, including rabbit anti-MST4 (Abcam; ab52491) and mouse anti-BrdU(GE Healthcare; RPN202), at 4°C overnight. The slides were then stained with a corresponding secondary antibody at 37°C for 20 min. Finally, the sections were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. Histopathology and immunohistochemistry analysis were performed independently by two pathologists without knowledge of the backgrounds of the patients. Staining intensity and percentage of staining-positive cells were evaluated for the IHC scores. Staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Percentage of stained cells were classified as 0 (<5 %), 1 (≥5%, <25 %), 2 (≥25, <75%) and 3 (≥75%). The final IHC score is the score of intensity multiplied by the score of percentage. The scores 0, 1, 2, 3, 4 were defined as low, and the scores 6 and 9 were defined as high. Slides with conflicting evaluations were reassessed, and a consensus was reached.

Tissue samples were fixed in 10% formalin, paraffin-embedded and cut in 3-μm sections, which were mounted in superfrostplus microscope slides (Thermo Scientific, Cat. 165061) and dried. The immunohistochemistry was performed using an automated immunostaining platform (Ventana discovery XT, Bond Max II, Leica). Antigen retrieval was performed with low pH buffer (CC1m) for p-H2AX and high pH buffer (ER2) for BrdU. Endogenous peroxidase was blocked (peroxide hydrogen at 3%), and slides were then incubated with anti-BrdU (BU-1, GE Healthcare, RPN202, Lot. 341585, 1:100) and phospho-histone H2AX (Ser139) (γH2AX, JBW301, Millipore, 05-636, Lot. DAM1493341, 1:4000). After the primary antibody, slides were incubated with the corresponding secondary antibodies when needed (rabbit anti-mouse Abcam) and visualization systems (Omni Map anti-Rabbit, Ventana, Roche; Bond Polymer Refine Detection, Bond, Leica) conjugated with horseradish peroxidase. Immunohistochemical reaction was developed using 3,30- diaminobenzidine tetrahydrochloride (DAB) (ChromoMap DAB, Ventana, Roche; Bond Polymer Refine Detection, Bond, Leica), and nuclei were counterstained with Carazzi’s hematoxylin. Finally, the slides were dehydrated, cleared, and mounted with a permanent mounting medium for microscopic evaluation.

Glioma cells were labeled with BrdU (GE Healthcare; RPN202) were performed as previously described^{44 (link)}.

Cell death was examined by TUNEL staining on slides using the ApopTag Peroxidase kit (Millipore). Mitotic cells were identified by antibody staining for phospho-histone H3 (PHH3, Cell Signaling) using a standard citrate buffer antigen retrieval and detection with a peroxidase conjugated secondary antibody. To track DNA synthesis, 10 mg/kg bromo-deoxyuridine (BrdU) was administered to pregnant dams by intraperitoneal injection two hours prior to harvesting. Briefly, sections were pre-treated with proteinase K, exonuclease III and DPN1, and BrdU was detected with the anti-BrdU antibody (RPN202; GE Healthcare) [35 (link)]. In each case, at least 1 section from 2 animals was counted per genotype. We counted the positive cells in each frontal bone primordia, and because it consists of ordinal data, it cannot be averaged. We found that the data fell naturally into two categories. With the PHH3 data, there were 8 sections that were below 6 positive cells, while the remaining sections were greater than 6 cells. For the TUNEL data, no wildtype section had greater than 3 apoptotic cells, therefore we analyzed the data using these categories.