Bond max autostainer system

The immunohistochemical staining was performed with Leica Microsystems Bond-Max Autostainer System as previously described [29 (link)]. The 5-µm thick specimen sections were deparaffinized at 60^oC using 60-minute incubation with the Bond Dewax Solution. The antigen retrieval was performed by incubating slides with the Bond Epitope Retrieval Solution 2 for 30 min at 100^oC, followed by 5-minute incubation with 3 % hydrogen peroxide.
Primary antibodies used were the rabbit polyclonal anti-SOX2 antibody (clone SP76, Cell Marque, Rocklin, CA) at 1:100 dilution and the mouse monoclonal ant-OCT4 antibody (clone sc-5279, Santa Cruz Biotechnology, Dallas, TX) at 1:300 dilution. The immunohistochemical procedure was performed using the Bond Polymer Refine Detection kit (Leica Microsystems), a 3-step indirect immunoperoxidase technique. The primary antibodies were applied for 40 min at room temperature, followed by 8 min incubation with the Post Primary Polymer and the Polymer Poly-HRP IgG, respectively. The sections were then incubated with diaminobenzidine for 4 min and counterstained with hematoxylin. The Bond Wash Solution was used to rinse between each step. As positive controls, tonsillar tissue (Fig. 1J) and germinoma (Fig. 1K) samples were used for SOX2 and OCT4 immunostainings, respectively. Negative controls were prepared using isotype-matched antibodies (Fig. 1L).

The immunohistochemical staining was performed with a Leica Microsystems Bond-Max Autostainer System according to manufacturer protocols. Arid1a (clone EPR13501–73, Abcam, dilution 1:1000), met (clone 4F8.2, Sigma Aldrich, dilution 1:250), pten (clone 138G6, Cell Signaling, dilution 1:100) and p53 (clone DO-1, Immunotech, dilution 1:50) were applied to consecutive 4-μm FFPE TMA sections. Appropriate positive and negative controls were run concurrently. Representative cases altered in p53, arid1a and met are shown in Supplementary Figure S3.

Areas with a high cellularity were selected for tissue microarrays. Immunohistochemical staining was performed as described previously [12 (link)]. E-cadherin (1:50 dilution; DAKO, Glostrup, Denmark; Catalogue No. M3612) and N-cadherin (1:500 dilution; Abcam, Cambridge, UK; Catalogue No. ab12221) antibodies were applied into a Bond-max autostainer system (Leica Microsystems, Bannockburn, IL, USA). Antigen retrieval was carried out using citrate buffer at pH 6.0. Negative controls were prepared without using primary antibodies.
All immunostained slides were evaluated twice by two independent observers (NMG and LKH) with no knowledge of the clinical details. E- and N-cadherin immunohistochemistry showed cytoplastmic positivity in glioma cells and sometimes stained the cytoplasmic borders. The intensity of staining was initially classified into 4 grades: 0, no immunoreaction; 1, weak positivity; 2, moderate positivity; and 3, strong reactivity. With N-cadherin staining, cases of grades 0 and 1 positivity were grouped as a low-expression, and cases of grades 2 and 3 as a high-expression for statistical convenience. Two pathologists re-evaluated cases with discordant staining intensity together and made concessions for such cases.