Anti ki 67 monoclonal antibody mib 1

The sections were incubated with primary monoclonal anti-LL-37 antibody (1:200; Santa Cruz Biotechnology, sc-166770, Dallas, TX). In addition, to determine the proliferative cell activity and its correlate with CAMP/LL-37 expression, we examined Ki-67 expression using anti-Ki-67 monoclonal antibody (MIB-1, Dako, Carpinteria, CA). The sections were incubated with primary antibodies at 4°C overnight after antigen retrieval by microwave treatment in citrate buffer (pH 6.0), detection by the avidin-biotin peroxidase complex system using an LSAB kit (Doko, Kyoto, Japan). A labeling index, percentage of CAMP/LL-37 or Ki-67 positive cells was determined by examining of at least 1000 tumor cells at 200× magnification. The expression of CAMP/LL-37 and Ki-67 were divided into high expression (more than 20% positive cells) and low expression (less than 20% positive cells).

Tumor tissue samples were collected from adult patients with GBM tumors (WHO grade IV) undergoing complete or partial surgical resection at the Institute of Neurosurgery, Catholic University School of Medicine in Rome. Informed consent was obtained from the patients before surgery. Experimental protocols were approved by the ethical committee of the Catholic University of Rome. Experiments were performed according to the guidelines approved by ethical committees of the Istituto Superiore di Sanità and Catholic University of Rome. Clinical and pathological features are summarized in Supplementary Table S3.
The expression of the proliferation marker Ki-67 and of Phosphatase and Tensin Homolog (PTEN) were characterized on tumor specimen by immunohistochemistry on deparaffinized sections using the avidin-biotin-peroxidase complex methods (ABC-Elite kit, Vector Laboratories), anti-Ki67 monoclonal antibody (MIB-1, Dako) and anti-PTEN mouse monoclonal antibody (clone 28H6; Novo Castra, Newcastle, United Kingdom). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation patterns were assessed on genomic DNA extracted from paraffin-embedded tissue by methylation-specific PCR as previously described15 (link). Levels of VEGF and EGFRvIII were assessed as previously described46 (link).

Formalin-fixed, paraffin-embedded tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) for tumor pathology. These sections were deparaffinized, rehydrated, and washed in phosphate-buffered saline. Endogenous peroxidase was quenched. A blocking step was included using 1% bovine serum albumin together with avidin and biotin blocking solutions. To determine the proliferative activity, Ki-67 immunostaining was performed using an anti-Ki-67 monoclonal antibody (MIB-1; Dako, Carpinteria, CA, USA). Apoptosis was measured by TUNEL assay using a commercially available apoptosis in situ detection kit (Wako Pure Chemical, Osaka, Japan). Visualization of the immunoreaction was performed with 0.06% 3,3′-diaminobenzidine (DAB) (Sigma Chemical, Atlanta, GA, USA). A dark accumulation of DAB in the nuclei was judged to indicate a positive reaction for TUNEL and Ki-67. The apoptotic index was calculated as the average number of TUNEL-positive cells in 10 areas at high power field (×400). The proliferation index was calculated as the average number of cancer cells with nuclei stained for Ki-67 in 10 areas at high power field (×400).