Ki 67 primary antibody

Immunohistochemistry was performed as previously described 39, 40. Ki‐67 primary antibody was obtained from Dako (Golstrup, Denmark). The paraffin‐embedded sections of the xenografts were detected using the TUNEL assay kit (R&D Systems, Minneapolis, MN, USA) for apoptosis analysis.

Human classical meningiomas (WHO grade I, n = 54), atypical meningiomas (WHO grade II, n = 28) and anaplastic meningiomas (WHO grade III n = 24) were provided by the Department of Neuropathology, Institute of Pathology, Changzheng Hospital and PLA NO.322 hospital. The immunohistochemistry was performed as previously described [17 (link)]. Briefly, Formalin-fixed, paraffin-embedded, 3-μm human tissue sections were incubated in nonimmune serum for 30 min and then incubated overnight at 4°C in RLIP76 primary monoclonal antibody(1:500; Abcam, ab56815) or Ki-67 primary antibody(1:75; Dako, Glostrup, Denmark). The staining intensity and percentage scores were measured as described [17 (link), 20 (link)]. For statistical analysis, RLIP76 expression was divided into “high” (++ and +++) vs. “low” (+ and-). Two independent pathologists examined 5 random fields (1 field = 0.159 mm2 at ×100 magnification) in each sample and scored each sample without knowing patient outcomes (double-blinded).

For immunohistochemistry, the tissue sections from untreated and treated tumour-bearing mice were deparaffinized, rehydrated, and washed. The tissue sections were then heat treated (95 °C) in antigen unmasking solution (Vector Laboratories, Burlingame, CA) for 30 min. Tissue sections were blocked in goat serum (1:10 in PBS) for one hour at room temperature, treated with Ki-67 primary antibody (rat monoclonal, Dako, 1:100) overnight at 4 °C followed by Alexafluor conjugated anti-rat secondary antibody at 1:100 dilution. Nuclei were stained using NucBlue (Invitrogen). Images were captured by the EVOS FL Cell Imaging System fluorescent microscope. For Iba-1, tissue sections were blocked with Iba-1 primary antibody (rabbit monoclonal, abcam, 1:1000) overnight at 4 °C followed by biotinylated anti-rabbit secondary at 1:2000 dilution. Avidin-biotin reaction was complete by incubating the tissues in ABC reagent (Vector Lab) for 30 min followed by DAB substrate reaction. Methyl green was used for staining the nucleus.