Ventana benchmark immunostainer

For the construction of tissue microarrays (TMA), 1 mm tissue cylinders were extracted from formalin-fixed and paraffin-embedded tumor samples archived in the department Neuropathology after evaluation and marking of the respective hematoxylin and eosin stains. For most of the cases, enough tissue was available to retrieve two tissue probes. A conventional microarrayer was used (Beecher Instruments, Sun Prairie, WI, USA). TMAs were cut with a microtome, and 4 μm tissue slices were produced and dried at 80° for 15 min. Immunohistochemical staining was done with a Ventana BenchMark immunostainer (Ventana Medical Systems, Tucson, AZ, USA).
Pretreatment with cell conditioning solution CC1 (pH 8.5) was done for 14 (CD68), 40 (MIB1, CD3 and CD163) or 64 min (CD8) followed by primary antibody incubation at 37° (CD8 (ready to use, Roche, Basel, Switzerland) and CD163 (1:1000, ABD Serotec, Puchheim, Germany)) or 42 °C (MIB1 (1:200, DAKO, Santa Clara, CA, USA), CD3 (1:500, Thermo Fisher Scientific, Waltham, MA, USA) and CD68 (1:200, Agilent DAKO, Santa Clara, CA, USA)). Subsequently, OptiView HQ universal linker was applied for 12 min, followed by incubation with OptiView HRP Multimer for 12 min. Counterstaining was done with hematoxylin for 4 min.
As controls, the human cerebral and cerebellar cortex, as well as a sample of a colorectal carcinoma metastasis, were placed on each TMA block.

Formalin-fixed, paraffin embedded 4 µm sectioned patient samples and mouse tumour xenografts were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc. Tucson, AZ, USA) as described previously [18 (link),28 (link)]. Tumour sections were de-waxed with Ventana EZ Prep and endogenous peroxidise activity blocked using the Ventana Universal DAB inhibitor. Primary antibodies against p-Src, t-Src, Ki67, CA125, Oct4, CD117 (c-kit), CD133 and CD31 were diluted as per manufacturer’s instructions. The sections were counter-stained with Ventana Haematoxylin and Blueing solution, and primary antibody staining was detected using the ultra-View Universal DAB detection Kit (Roche, Basel, Switzerland). Negative controls were prepared by incubating each tumour sections without primary antibodies. Immunohistochemistry images were taken using an Evos FL Auto 2 microscope (Thermo Fisher Scientific). DAB staining (5× 200 µm images/section) was quantified using processing packages in ImageJ (positive pixels or number of positive cells) and Aperio (strong positive staining pixel count). Quantification was standardised to area and normalised to the negative control sections. Results displayed as mean ± SD.

Archived formalin-fixed and paraffin-embedded tissue samples were used for the extraction of two biopsy punches of 2 mm diameter each to construct tissue microarrays with a conventional tissue microarrayer (Beecher Instruments, Sun Prairie, WI, USA). Tumor cylinder extraction was done after evaluation of H&E slides for suitable areas. The newly formed tissue blocks were cut into 4 μm slices with a microtome. After subsequent drying at 80°C for 15 min, subsequent immunohistochemical staining for H3K27me3 (1:200, rabbit monoclonal antibody C36B11, Cell Signaling, Danvers, MA, USA) and EZH2 (1:100, rabbit monoclonal antibody D2C9, Cell Signaling, Danvers, MA, USA) were done with a Ventana BenchMark immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Analysis of immunohistochemical staining were scored independently by two pathologists.

Hemotaxylin & Eosin (H & E) staining of mouse organs and tumors was performed by the staff at the Anatomical Pathology Laboratory Services at The Royal Children's Hospital, Melbourne, Australia according to the standard H&E protocol [57 (link)]. Immunohistochemistry analysis of mouse tumors was performed on formalin fixed, paraffin embedded 4 μm sections of the xenografts and were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA) as described previously [3 (link), 20 (link)]. Negative controls were prepared by incubating each tumor section without primary antibodies. Sections of human breast tissue, high-grade ovarian tumors and human tonsils were used as positive controls to determine the staining efficacy of primary antibodies. Immunohistochemistry images were taken using an Aperio ImageScope (Leica Microsystems, Mt Waverly, Australia) with the associated digital pathology viewing software. DAB staining was measured using the open source image processing package Fiji (https://fiji.sc/) with a plug-in developed to recognize DAB staining for 10 randomly captured images per section. DAB staining intensity of the negative control was subtracted from the DAB staining of the antibody of interest to get a measure of DAB staining of interest.

For immunohistochemistry, formalin fixed, paraffin embedded 4 μm sections of the xenografts were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA) previously [45 (link)]. Immunohistochemistry images were taken using Axioskop 2 microscope, captured using a Nikon DXM1200C digital camera and processed using NIS-Elements F3.0 software. Images were scored independently by four reviewers blind to the molecular data as previously described [46 (link)].

Immunostaining for MBOAT7 was performed on liver biopsies of CHC patients. Formalin-fixed, paraffin-embedded 4 μm sections were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA). Slides were incubated with a dilution of 1:10 of rabbit polyclonal antibody (Ab) specific for Human MBOAT7 (SIGMA ALDRICH). Negative controls where the primary antibody was excluded confirmed the specificity of immunostaining. Detection was performed using Ventana's Ultra View DAB kit (Roche/Ventana 05269806001). The pathologist (D.M.) evaluated the MBOAT7 immunostaining semi-quantitatively in a blinded fashion regarding any of the histological and clinical characteristics of the patients. The extent of staining was scored according to its amount and intensity using a 4-point scoring system as follows: 0=no staining; 1=positive staining in <33% of cells; 2=33–66% of positive cells; and 3=positive staining in more than 66% of cells.

Available data from immunohistochemical staining for COX2 and MIB1 according to our prior study were used [1 (link)]. Tissue microarrays (TMA) were constructed with 2 representative 1 mm tissue probes for each tumor using a conventional microarrayer (Beecher Instruments, Sun Prairie, Wisconsin, USA). With microtomy 4 μm slices were produced and dried at 80° for 15 min. A Ventana BenchMark immunostainer (Ventana Medical Systems, Tucson, Arizona, USA) was used for immunohistochemical staining of COX2 (1:800, Biozol, Eching, Germany). Quantification was done by semiquantitative assessment and formulation of an intensity distribution score (ID-score): 0 = 0–5%, 1 = 5–25%, 2 = 25–50%, 3 = 50–75%, 4 = 75–100%). MIB1 expression was analyzed via quantification on digitalized whole section stainings that were done within the routine neuropathological workup.