Immunohistochemical Characterization of Glioblastoma

Besides haematoxylin and eosin (H&E) staining, IHC was performed using a Ventana Full BenchMark^® XT automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). The ultraView^TM Universal DAB Detection Kit (Ventana) was the detection system. Heat-induced epitope retrieval was obtained with cell conditioning solution (CC1-Tris based EDTA buffer, pH 8.0, Ventana). Negative controls were obtained by the omission of the primary antibody. The primary antibodies are listed in Table 2. Nestin, Sox2, and MSel1 were considered as representative of GB stem cells (GSCs)/progenitors [73 (link),74 (link)].
Double immunostainings for GFAP/Nestin, GFAP/ATRX, and CD34/Iba-1 were performed with the ultraView^TM Universal Alkaline Phosphatase Red Detection Kit (Ventana).
NG2/CSPG4’s immunoreactivity was evaluated using a semi-quantitative system for the percentage of positive cells, the staining intensity, and type of distribution (focal or diffuse) in five randomly selected microscopic high-power fields (HPF), at a ×400 magnification, per tumor section.
The frequency of glioma-associated microglia/macrophages (GAMs) was quantified as previously described [75 (link)].
IHC for ATRX was used as a surrogate marker for the mutation status of the ATRX gene. The quantification methods for ATRX have been already reported [76 (link)].