Ultraview universal dab detection kit

Formalin-fixed paraffin-embedded tumor samples were subjected to immunocytochemistry according to the manufacturer’s antibody protocol. Samples were developed either by using either secondary antibodies linked to horseradish peroxidase or secondary antibodies linked to a fluorophore. Immunostaining was performed on 4 µm sections from formalin-fixed paraffin-embedded tissues. Staining was performed either manually or on the automated immunostainer Beckmarck XT (Ventana Medical Systems, Roche, Tucson, AZ, USA). Antibodies were visualized by the UltraView^TM Universal DAB Detection Kit (Ventana Medical Systems). For samples processed manualy antigen retrieval was performed using target retrieval solution pH 6.0 (Dako,Agilent, Santa Clara CA, USA)) Samples were scanned (panoramic slide digital scanner) and evaluated by two independent pathologists (using 3DHistech software).

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)].

Microsatellite instability (MSI) testing was performed in 14 cases using a fluorescent PCR-based assay (MSI Analysis system, Version 1.2, Promega, Madison, WI). Briefly, this test assessed 5 mononucleotide repeats (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeats (PentaC and PentaD) on genomic DNA and matched normal, where available. The fluorescently labeled PCR products were analyzed by capillary gel electrophoresis. MSI was determined if the tumor alleles showed a size difference ≥3 bp. Tumors with 2 or more microsatellite unstable markers were classified as MSI-H; the remainder were classified as microsatellite stable (MSS). Immunohistochemistry was performed for MLH1 (clone M1, predilute, Ventana), MSH6 (clone 44, predilute, Ventana), MSH2 (clone G219-1129, predilute, Cell Marque) and PMS2 (clone EPR3947, predilute, Cell Marque) using an autostainer (Benchmark Ultra^TM Ventana, Tuscon, AZ) after heat induced antigen retrieval (EDTA, pH 7.3). The ultraView^TM Universal DAB Detection kit (Ventana) was used for visualization. Absence of nuclear staining was evaluated in lesional tissue; adjacent stromal cells were used as an internal control.
Methods for DNA extraction, immunohistochemistry, flow cytometry, and Sanger sequencing are described in the supplemental methods.

For IC analysis, SKMel103, SKMel147, SKMel28 and UACC903 cells were seeded directly on cover slides in a 24-well plate (Sarstedt, Nümbrecht, Germany) at 50% confluence, incubated overnight and subjected to treatment the next day. Cell staining was performed as previously described^{54 (link)}. For IF and IHC, 4 µm sections of formalin-fixed paraffin-embedded tumor samples were subjected to immunocytochemistry according to the manufacturer’s antibody protocol. The samples were developed by using either secondary antibodies linked to horseradish peroxidase with the UltraView^TM Universal DAB Detection Kit (Ventana Medical Systems) or secondary antibodies linked to fluorophores. Staining was performed either manually or on the automated immunostainer Beckmarck XT (Ventana Medical Systems, Roche, Tucson, AZ, USA). For the samples processed manually, antigen retrieval was performed using target retrieval solution pH 6.0 (Agilent). The samples were scanned (panoramic slide digital scanner) and evaluated by two independent pathologists (using 3DHistech software).

Formalin-fixed paraffin embedded (FFPE) tumor samples were subjected to immunocytochemistry according to the manufacturer's antibody protocol. The samples were developed using secondary antibodies conjugated with horseradish peroxidase and diaminobezidine as a substrate. Immunohistochemical staining was performed on 4 μm sections from formalin-fixed paraffin-embedded tissues. Melan-A, HMB-45 and Ki67 were obtained from Master Diagnostica (Granada, Spain); c-KIT, p-S6 and p-ERK1/2 were purchased from Cell Signaling (Danvers, MA, USA). The paraffin sections were automatically de-paraffinized and treated with cell conditioning 1 solution (pH8) for antigen retrieval (Ventana Medical Systems, Tucson, AZ, USA). Staining was performed with an automated immunostainer (Beckmarck XT, Ventana Medical Systems). Antibodies were visualized using the ultraViewTM Universal DAB detection Kit (Ventana Medical Systems). The samples were evaluated by two independent pathologists.