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14 protocols using ventana ultra

1

Immunohistochemical Analysis of 3D Cell Spheroids

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Spheroids were collected and pooled, washed in PBS, and fixed in formalin for 1 h at room temperature. After washing twice in PBS, spheroids were transferred to a cryomold and suspended in Histogel, before paraffin embedding and sectioning (4 μm). Cellular morphology was assessed via hematoxylin and eosin (H+E) staining, and immunohistochemistry was performed using the Ventana Ultra and associated reagents from Roche. Details of the conditions for each of the antibodies used are reported in Table 3.

Summary of antibodies and conditions used for immunohistochemistry on the Ventana Ultra

AntibodySupplierHostPretreatmentDilution
CD68NovocastraMousemCC1, pH81:100
α-SMADakoMousemCC1, pH81:100
COL1A1BiositeRabbitstdCC1, pH81:1000
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2

Neuroblastoma Tumor Immunophenotyping

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Diagnostic neuroblastoma samples from the Children’s Oncology Group (COG) and Stanford University, School of Medicine, Department of Pathology were used. After pathology review according to the protocol of the COG Neuroblastoma Biology Study, unused sections were available for immunostaining. Those cases were filed at the COG Neuroblastoma Pathology Reference Laboratory, which were identified only by the COG accession number and not associated with patient information. IRB approval of the cases was obtained at the time of study enrollment by the contributing institution. Neuroblastoma specimens archived at Stanford University were used to perform IHC staining on serial sections of the tumor tissues. Formalin-fixed paraffin embedded tumor sections were subjected to IHC analysis. Clone KP1 for CD68 (Dako) and clone MRQ-26 for CD163 (Ventana/Cell Marque) were used to stain the corresponding antigens. The automated IHC processor was used to process tumor sections according to the manufacturer’s instructions (Ventana Ultra, Roche). Immunohistochemical evaluation was performed in the representative and viable tumor areas away from necrosis and fibrosis.
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3

Automated BRAF V600E Immunostaining Protocol

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Automated immunostaining was performed on the Ventana Ultra (Roche). The protocol involved dewaxing with Ezprep solution, followed by heat-induced epitope retrieval with CC1buffer for 64 min and then endogenous peroxidase inhibition. Slides were then incubated with the ready to use BRAF V600E mutation-specific monoclonal antibody, (Ventana, clone VE1 (CE-IVD)) for 16 min at 37 °C. Chromogenic detection was carried out using the OptiView DAB IHC kit (Roche) along with an Optiview amplification kit (Roche). Four minutes Optivew amplification H2O2 and four minutes Optiview amplification multimer incubation times were used. Slides were counterstained with haematoxylin II (Roche) for 4 min followed by bluing reagent (Roche) for 4 min.
Positive staining was seen as the presence of unequivocal cytoplasmic granular staining of any intensity in the tumour cells. Negative staining showed the absence of cytoplasmic staining in the tumour cells.
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4

Tissue Immunohistochemistry Protocol

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Immunohistochemistry was performed using a Ventana Ultra (Roche Inc., Basel, Switzerland) steam-induced epitope retrieval platform on positively charged slides cut from formalin-fixed paraffin-embedded tissue blocks. Slides were labeled with the listed antibodies, which were previously titrated and validated for clinical diagnosis on paraffin embedded-human surgical specimens.
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5

Histological and Immunostaining Analysis of Lesions

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The biopsies of the lesions and adjacent skin were fixed in formalin and embedded in paraffin. Formalin-fixed paraffin-embedded tissue sections (4 µm) were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains (Sigma-Aldrich) according to the manufacturer’s protocol. Immunostainings were performed using a Ventana Ultra automated immunostainer (Ventana Medical Systems, Tucson, AZ). Stained cells were counted blindly. Refer to supplemental material for the list of antibodies (Supplementary Table 1).
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6

Automated Immunohistochemistry Staining

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Slides were processed for immunohistochemistry using Ventana Ultra (Ventana) or Dako Omnis (Agilent) immunohistochemistry systems. Antigen retrieval, staining with primary and secondary antibodies and washes were performed as part of automated programmes optimised for each antibody. Control tissue was stained in parallel on every slide. Details for each antibody are given in supplementary material, Table S1.
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7

Adipose Tissue Amyloid Characterization

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The adipose tissue aspirate was pressed overnight (10 kg), fixed in 4% neutral buffered formaldehyde and stained for congo red as described.21 The cover slips were removed without manipulation by incubation in xylene‐acetone until the coverslips fell off spontaneously. Afterwards, the samples were pretreated with pepsin solution (1 h, 37 °C, Zytomed Systems, Berlin, Germany) and afterwards immunostained with polyclonal rabbit anti‐ATTR (clone TIE, amYmed, Munich, Germany) at a dilution of 1:10 for 120 min using an automated platform (Ventana ULTRA; Ventana Medical Systems, Tucson, AZ 85755) with DAB chromogen.
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8

PD-L1 and MDM2 Expression in FFPE Tissues

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Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections on the Ventana Ultra automated staining System (Ventana Medical Systems, Oro Valley, AZ, USA) using Ventana reagents, according to the manufacturer´s protocol. All cases were stained with an antibody against PD-L1 (clone 22C3, order no. M3653, 1:50, Dako, Cambridge, UK), MDM2 (clone 3G187, order no. 113-0230, 1:25, Zytomed, Berlin, Germany). Primary antibody detection was performed using the OptiView DAB IHC detection kit (Ventana). Appropriate positive and negative controls were used to confirm the adequacy of the staining. PD-L1 expression was evaluated using the MEL Score (PD-L1 positive tumor cells + PD-L1 positive mononuclear inflammatory cells/total tumor cells + total mononuclear cells). We classified PD-L1 staining into two groups: MEL score <1 and ≥1. MDM2 expression was specified as percentages of positive tumor cells. Images were acquired with a Zeiss Axioskop 2 plus microscope equipped with a Jenoptik ProgRes C10 plus camera and software (Laser Optik System, Jena, Germany).
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9

PD-L1 Immunohistochemistry Analysis in Cancers

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The immunohistochemistry reaction was performed using BenchMark Ventana Ultra™ (Ventana, Tucson, AZ, USA) platform, through multimer linked to horse radish peroxidase, to detect PD-L1 protein, as previously reported [25 (link)]. The anti-PD-L1 (E1L3N®) XP® Rabbit mAb, Cell Signaling Technology, was used as primary antibody and we used the OptiView DAB IHC Detection Kit, following manufacturer’s guidelines. Placental syncytiotrophoblast was used as positive control tissue. The combined positive score (CPS) was used to measure the expression of PD-L1. CPS corresponds to the ratio between the total of PD-L1 positive cells (tumor cell, lymphocytes and macrophages) and the total of viable tumor cells, multiplied by 100 [26 (link)]. Considering the experience of appropriateness of CPS cutoff in other types of tumors and no agreement of CPS cutoff for CUPs, we used CPS ≥ 1 in our study [27 (link)].
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10

Pathologic Tumor Assessment and PD-L1 Staining

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Pathologic tumor assessment and PD-L1 stainings were carried out in cooperation with the Institute of Pathology, MHH, Germany. Selected samples included the worst differentiation and the rim of necrosis for the primary tumor and cava thrombus, if possible. Immunostaining of 3–5 μm formaldehyde-fixed, paraffin-embedded tissue sections was routinely performed on an automated platform (Ventana ULTRA, Ventana Medical Systems, Tucson, AZ, USA) using a PD-L1 antibody (clone 22C3, 1:40, Agilent, Santa Clara, CA, USA) after heat-induced epitope retrieval with CC1 solution (Ventana Medical Systems). Positive controls (tonsil tissue) were included in all staining procedures.
The evaluation of the following scores was carried out by a trained pathologist, Dr. Jan Hinrich Bräsen (JHB). These scores have been comprehensively described in the literature [10 (link), 12 (link)]. CPS=Number of stained tumor cells+Number of stained mononuclear inflammatory cellsTotal number of tumor cells×100 IC - score\%=Stained immune cellsTumor area TPS\%=Number of stained tumor cellsTotal number of tumor cells
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