We also reviewed the molecular basis of Fas-mediated apoptosis in malignant gliomas. Glioblastoma specimens from 97 patients who had not been previously treated were retrieved from the archives of the Departments of Pathology at São Paulo Federal University (n = 60) and Ribeirão Preto Medicine Faculty

at São Paulo University (n = 37). The tumor specimens were re-examined and confirmed to be glioblastomas according to the criteria of the most recent WHO Classification of Central Nervous System Tumors [22]. All of the patients had undergone surgery during the 15-year period from 1992 through 2006. This study was approved by the Ethics Committees of both institutions (Resolution No. 196 of Brazilian National Health Council). Histological sections (4 μm) were cut from each tissue block, Talazoparib purchase stained by hematoxylin–eosin, and carefully reviewed by 3 independent pathologists. The areas most representative of each tumor were selected

for analysis. Cylindrical cores were removed and used in the construction of tissue microarray (TMA) blocks. Five TMA blocks were constructed using a Beecher tissue array instrument™ (Beecher Instruments, Silver Spring, MD, USA), according to the manufacturer’s instructions, in the following stages: (1) Two different areas of the tumor were marked in the original donor block for sampling (necrotic zones and perinecrotic palisading cells were not included in the samples), (2) cylindrical holes were created in the receptor block using the TMA platform. Positions were created in the receptor Selleck GSK 3 inhibitor blocks and were separated by approximately 500 μm such that a matrix of holes for the tissue samples was created, (3) 1-mm diameter cylinders of tissue were extracted from the areas of interest in the donor blocks using a 1-mm-diameter needle (TMArrayer Punch Beecher Instruments™), ID-8 (4) the cylindrical tissues obtained from the donor blocks were

transferred to the holes in the receptor blocks, and (5) finally, the quality of the blocks (representativeness of the tumor samples) was assessed before storage. Twenty-five control cores obtained from normal brains harvested from 25 autopsied patients (6–12 h postmortem) were included as controls. The immunohistochemical procedures were performed on 4-μm-thick sections that were obtained from the TMA blocks and mounted on slides pretreated with 3-minopropyl-triethoxysilane (Sigma). To aid in the adhesion of the slices from the TMA blocks to the silane-treated slides, an adhesive tape system (Instrumedics Inc., Hackensak, NJ, USA) was also used. Briefly, for immunostaining, the slides were deparaffinized, and rehydrated through a graded ethanol series. For antigen retrieval, slides were placed in a 0.01 M citrate buffer (pH 6.0), heated in a steam bath for 3 min, and allowed to cool at room temperature for 30 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 15 min, followed by washing in 0.05 M Tris buffer (pH 9.5).