The slides were again placed in phosphate buffered saline (0.01 M PBS [pH 7.4]) and allowed HIF inhibitor to cool at room temperature for 30 min. All of the immunomarkers that were evaluated were examined on slides that underwent treatment for antigen retrieval. The endogenous biotin was blocked using 0.02 M PBS/0.3% Triton X100 (pH 7.4) and

5% skim milk for 4 h at room temperature. Incubation with anti-FasL rabbit polyclonal antibody (C-178, 1:500; Santa Cruz Biotechnology), anti-Fas rabbit polyclonal antibody (FL-335, 1:200; Santa Cruz Biotechnology), anti-cleaved caspase-8 mouse monoclonal antibody (AP1013, 1:100; Calbiochem), anti-cleaved caspase-3 rabbit polyclonal antibody (AP1027, 1:500; Calbiochem), anti-IDH1 rabbit polyclonal antibody (AP7454a, 1:50; Abgent), or anti-MGMT mouse monoclonal antibody (SPM287, 1:150; Santa Cruz Biotechnology) diluted in PBS with 1.0% bovine serum albumin (Sigma, USA) lasted for 12 h in a moist chamber at 4 °C. The slides were then washed in PBS and incubated

with secondary biotinylated antibody followed by streptavidin–biotin-peroxidase (anti-mouse or anti-rabbit Kit LSAB, DAKO) for 30 min each. Finally, to visualize the reactions, the slides were incubated with light-sensitive 3,3′-diaminobenzidine tetrahydrochloride (Sigma) in 0.05 M PBS (pH 7.6) and quickly FDA approved Drug Library counterstained with Harris hematoxylin. Coverslips were applied using Entellan (Sigma). A positive reaction was visualized as a brown deposit in the cell that indicated an area where the antigen–antibody reaction had occurred. Negative and positive controls were run simultaneously. Lymphoid tonsil tissue with follicular germinative centers was used as a positive control for FasL, Fas, cleaved caspase-8, Ceramide glucosyltransferase and cleaved caspase-3.

Placenta and normal colon, which had immunohistochemistry performed separately from the TMAs, were used as positive controls for IDH1 and MGMT, respectively. Negative controls consisted of slides that underwent the same procedure, except the incubation with primary antibody was eliminated. The staining patterns were analyzed according to their distribution and intensity, and the pathologists were blinded to the clinicopathological data of the GBM patients. A numerical scoring system consisting of 2 categories was used to assess FasL, Fas, cleaved caspase-8 and cleaved caspase-3 expression. Category A documented the number of immunoreactive cells (only ones with their respective nuclei inside were counted) as follows: 0 or negative (no immunoreactive cells or <10% immunoreactive cells), 1 (≥10% and <50% immunoreactive cells), or 2 (≥50% immunoreactive cells). Category B documented the intensity of the immunostaining as follows: 0 or 1 (no immunostaining or weak staining, respectively) or 2 (moderate or strong). The values for categories A and B were summed to provide an “immunoreactivity score”, which could range from 0 to 4.