Automated immunofluorescence (IF) was performed on de-paraffinised 3 μm FFPE tissue sections using a Dako link 48 instrument (Dako, Agilent Technologies). The primary antibody against Pan-cytokeratin (PanCK; Cat# Z0622, Agilent Technologies) was utilised to visualise urothelial cells and nuclei were counterstained with Hoechst (Hoechst 33342, Cat# H3570, ThermoFisher Scientific). A Carl Zeiss AxioScan.Z1 whole slide scanner (Zeiss, Göttingen, Germany) was used to capture and digitise whole slide fluorescence images with a 20x objective (see detailed immunofluorescence and digitisation protocol in the Supplementary Material).
Dako link 48
Manufactured by Agilent Technologies
Sourced in United States
The DAKO Link 48 is an automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining system. It is designed for the efficient and reliable processing of tissue samples in a clinical laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Pt link, by Agilent Technologies (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Envision flex detection kit, by Agilent Technologies (1 mentions)
Haematoxylin, by Agilent Technologies (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Dm828, by Agilent Technologies (1 mentions)
Bond rxm, by Leica (1 mentions)
Dako omnis, by Agilent Technologies (1 mentions)
7 protocols using dako link 48
1
Automated Immunofluorescence Imaging of Urothelial Cells
2
Immunohistochemical Analysis of OLFM4 in Human Tissues
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Immunohistochemistry was performed on archival paraffin embedded human tissue obtained from normal colon, duodenum, lung, thymus, liver, spleen. Six needle biopsies from the prostate gland were included. Two needle biopsies contained only malignant tissue, while the remaining four contained both benign tissue and adenocarcinoma. Gleason score was between 6 and 9. Furthermore, different subtypes of colonic adenocarcinomas and colonic adenomas/precursor lesions were included. Tissue material had been fixed in buffered formalin and embedded in paraffin. Three micron sections were made. Immunochemical staining was done using the following murine antibodies as primary antibody OLFM4 #40 (0.48 μg/ml), OLFM4 #49 (0.70 μg/ml). The sections were pre‐treated in PT Link (Dako, Glostrup, Denmark) using high pH target retrieval solution (DM828; Dako) and stained in a DakoLink 48 (Dako) utilizing the Envision Flex+ detection kit (K8002; Dako). Primary antibodies were diluted in Antibody Diluent (DM830; Dako) and incubated for 20 min. The sections were counterstained with haematoxylin (Dako) and analysed using an Olympus BX51 microscope (PlanApo 20x/0.70 objective) with an Olympus UC30 camera and the Olympus cellSens software package (Olympus, Ballerup, Denmark).
3
Comparative Evaluation of PD-L1 IHC Assays
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Serial sections (3 mm) of the Melbourne TMAs were stained on the Dako Link 48 platform for Dako pharmDx kits for clones 22C3 (SK006) and 28-8 (SK005) and on the Ventana Benchmark Ultra platform for SP263 (790-4905) and the predilute FDA-approved SP142 assay kit (740-4859) according to the package inserts (Supplementary Table 2). Formalin-fixed, paraffinembedded agar cell pellets prepared from the PD-L1positive NCI-H226 cell line and human placental and tonsil tissues were used as controls.
To compare the performance of the 22C3 antibody on the different automated staining platforms, predilute 22C3 antibody (using the stock concentration provided in the Dako pharmDx PD-L1 kit) was run on the Ventana Benchmark Ultra system according to the standard Ventana protocols recommended for SP263 using the Opti-View HRP Multimer detection system (see Supplementary Table 2). All optimization protocols were performed simultaneously to limit machine, reagent, and run-to-run variation. TMA sections stained according to package instructions for 22C3 on the Dako Link 48 platform were compared with sections stained using the aforementioned protocol for 22C3 on the Ventana Benchmark Ultra system (hereafter referred to as 22C3-V), performed on the Melbourne TMAs at the Peter MacCallum Cancer Centre and on the Sydney TMAs at Royal Prince Alfred Hospital.
To compare the performance of the 22C3 antibody on the different automated staining platforms, predilute 22C3 antibody (using the stock concentration provided in the Dako pharmDx PD-L1 kit) was run on the Ventana Benchmark Ultra system according to the standard Ventana protocols recommended for SP263 using the Opti-View HRP Multimer detection system (see Supplementary Table 2). All optimization protocols were performed simultaneously to limit machine, reagent, and run-to-run variation. TMA sections stained according to package instructions for 22C3 on the Dako Link 48 platform were compared with sections stained using the aforementioned protocol for 22C3 on the Ventana Benchmark Ultra system (hereafter referred to as 22C3-V), performed on the Melbourne TMAs at the Peter MacCallum Cancer Centre and on the Sydney TMAs at Royal Prince Alfred Hospital.
4
High-Throughput Protein Microarray Immunostaining
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Briefly, a 2470 Arrayer (Aushon BioSystem, MA, USA) created a sample array on Oncyte Avid nitrocellulose-coated slides (Grace Bio-Labs, OR, USA). Immunostaining was performed on an automated slide stainer (Dako Link 48 – Agilent Technologies, CA, USA). Full details are provided in Supplementary Methods and raw data is provided in Supplementary Table S2 .
5
Immunohistochemical Evaluation of Glioma Markers
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Immunohistochemistry for ASCL1 was performed using a mouse monoclonal antibody (BD Bioscience, #556604, 1:100, high pH antigen retrieval) on a Bond RXm (Leica Biosystems) or a DAKO Link48 (Agilent) automated staining system at two different institutions (Children's Hospital Los Angeles and University Hospital Münster). Samples stained on both platforms in parallel for validation yielded comparable results. Immunohistochemical staining for GFAP (#GA524, Agilent), OLIG2 (#18953, Immuno-Biological Laboratories, Inc.), and synaptophysin (#M7315, Agilent), was performed using the streptavidin–biotin method on an automated staining system (DAKO OMNIS, Agilent). For the purpose of the present study, immunohistochemical staining results were rated as absent, focal (< 5% of tumor cells) and present (≥ 5% of tumor cells).
6
Multimarker IHC Staining Protocol
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IHC was performed on FFPE sections of glass slides. Slides were stained using the Agilent DAKO Link 48 (Santa Clara, CA, USA) automated platform and staining techniques, per the manufacturer’s instructions, and were optimized and validated per CLIA/CAP and ISO requirements. Staining was scored for intensity (0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining) and staining percentage (0–100%). Positive expression of immune cell (IC) PD-L1 (SP142), tumor cell ESTROGEN RECEPTOR (ER), and tumor cell PROGESTERONE RECEPTOR (PR) was defined as ≥1+ stain intensity and ≥1% of cells stained. Positive HER2 expression was determined according to the 2018 ASCO-CAP guidelines (8 (link)).
7
PD-L1 Immunohistochemistry Staining Protocol
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Immunohistochemistry (IHC) for PDL1 was performed on FFPE sections of glass slides. Slides were stained using the Agilent DAKO Link 48 (Santa Clara, CA, USA) automated platform and staining techniques, as per the manufacturer’s instructions, and were optimized and validated per CLIA/CAP and ISO requirements. Staining was scored for intensity (0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining) and staining percentage (0–100%). PD-L1 (SP142)-positive staining was defined as ≥2+ and ≥5% tumor cells.
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