Benchmark xt immunostainer

Diffuse astrocytomas with IDH1^R132H mutation (n=3) or wild-type IDH1/2 (n=3) were processed for immunohistochemistry. IDH1/2 wild-type status was confirmed by Sanger sequencing. Four-micrometre thick sections were cut from the selected formalin paraffin embedded specimens and stained using a Benchmark XT immunostainer (Ventana). The Ventana staining protocol included a pretreatment with Cell Conditioner 1 for 40 min, followed by incubation with the primary antibodies at 37 °C for 60 min. All the antibodies were diluted in the Ventana dilution buffer as followed: p-S6^S240/244 (D68F8, Cell Signaling Technology, 1:1,000); p-AKT1^T308 (Novus Biologicals, 1:30). Incubation was followed by detection using Optiview DAB IHC detection kit (Ventana).

3–5 µm tissue sections of formalin‐fixed and paraffin‐embedded (FFPE) tissue were used. Immunostainings were performed on the BenchMark XT immunostainer (Ventana Medical Systems), using CC1 mild buffer or Ultra CC1 buffer (Ventana Medical Systems) for 30 min at 100°C, and using antibodies rabbit anti‐TFF3 (1:250, Abcam, ab108599), mouse anti‐FABP1 (1:1,000, Abcam, ab7366), rabbit anti‐OLFM4 (1:100, Atlas Antibodies, HPA077718), mouse anti‐EPCAM (1:100, Thermo Scientific, MS‐144‐P1), rabbit anti‐Ki67 (1:400, Abcam, ab16667), mouse anti‐Ki67 (1:50, Dako, M7240), rabbit anti‐LYZ (1:1500, Abcam, ab108508), rabbit anti‐EREG (1:50, Thermo Fischer Scientific, PA5‐24727), anti‐PARP1, mouse anti‐MUC2 (1:50, Leica, NCL‐MUC‐2), mouse anti‐CK17 (1:10, Dako, M7046), and mouse anti‐MMP7 (1:100, Thermo Fisher Scientific, MA5‐14215). Images were taken using AxioVert.A1 (Zeiss) or CQ1 (Yokogawa) microscopes or scanned using the Pannoramic SCAN 150 scanner (3DHISTECH).

Firstly, we purchased glioma tissue microarrays (GL1001a) from Biomax, Inc. (https://www.biomax.us/), which contained the tissue sections and clinical information. All human tissue specimens were obtained with the informed consent of donors through US Biomax, Inc. This tissue array included 70 cases of adult glioma (7 for Grade 1; 45 for Grade 2; 10 for Grade 3; 8 for Grade 4) and 8 normal brain tissue (Additional file 1: S1E). The core measured 1.5 mm in diameter with 5 µm tissue thickness. In the immunohistochemistry, the Ventana Benchmark^®XT immunostainer was used to perform immunohistochemical staining, ensuring consistency. Before staining, samples were heated in a pressure cooker for 30 min at 125 °C for antigen retrieval (0.01 M sodium citrate, pH 6.2), then washed three times in phosphate-buffered saline (PBS) for five minutes each. Next, the slides were uploaded to the autostainer according to the instructions from the manufacturer. The primary antibodies used here include AUP1 (Atlas antibodies #HPA007674), P53 (cell signaling # 2524), and KI67 (Abcam #ab15580). The secondary antibodies used Roche Diagnostics OptiView DAB IHC Detection Kit. Positive and negative controls were used to evaluate the antibodies' binding abilities. Robust staining of the positive control was found, but not that of the negative control.

Immunohistochemical staining was performed using the Immunostainer BENCHMARK XT (Ventana medical systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Anti-Ki-67 (MIB1, Dako, 1:200) was used as the primary antibody. Briefly, 4 µm-thick sections of paraffin-embedded tissue were transferred onto poly-l-lysine coated adhesive slides and dried at 62 °C for 30 min. After epitope retrieval, the samples were incubated with primary antibody followed by biotinylated secondary antibody, and signals were developed using peroxidase-labeled streptavidin and 3,3′-diaminobenzidine (DAB). Slides were counterstained with Harris’ hematoxylin. Positive control samples were stained with each batch.

Five-micrometer thick tumor sections were stained with anti-human P2X7 (Abcam, Cambridge, UK, 1:100 dilution) antibody in an automated immunohistochemistry processor (Immunostainer Benchmark XT; Ventana and Tuckson). For the detection of P2X7, sections were cut from formalin-fixed, paraffin-embedded blocks, deparaffinized with xylene, and rehydrated by sequential passages through decreasing (from 100% to 80%) concentrations of ethanol. Endogenous peroxidase activity was blocked by a 30-minute incubation at room temperature with methanol containing 3% H₂O₂. Tissue sections were then incubated at 98°C for 40 minutes in Target retrieval solution pH 9.0 (Dako); after several rinses in wash buffer (Dako), tissue sections were incubated overnight at 4°C with rabbit polyclonal anti-P2X7 antibody (ABCAM, 1:100). After incubation with the primary anti-antibody, tissue sections were rinsed twice in PBS and incubated for 30 minutes at room temperature with Dako Envision System HRP-conjugated rabbit antibodies. Tissue sections were then washed in PBS, and peroxidase activity was detected by incubation for 6 to 10 minutes at room temperature with Dako Liquid diaminobenzidine (DAB) Substrate Chromogen System (Dako). Counterstaining was conducted with Mayer's hematoxylin (Sigma-Aldrich).