Formalin-fixed paraffin-embedded (FFPE) blocks for 456 patients with sequencing data were sectioned and stained for anti-CD3 (clone 2GV6, predilute; Ventana Medical Systems), anti-CD4 (clone SD35, predilute; Ventana Medical Systems), anti-CD8 (clone SD57, predilute; Ventana Medical Systems) and anti-PD-L1 (clone SP263, predilute; Ventana Medical Systems) using an automated immunostainer (Ventana BenchMark ULTRA; Ventana Medical Systems, Tucson, AZ). Stained slides were digitised using an Aperio AT2 whole slide scanner. CD3, CD4, and CD8 staining were quantified using the Aperio Positive Pixel digital pathology tool (v9 algorithm at 0.16 colour saturation). PD-L1 expression was determined using the Combined Positive Score system (0: no stain (negative); 1: ≥1% positive tumour cells staining).
Anti cd3 clone 2gv6
Manufactured by Roche
Sourced in United Kingdom
Anti-CD3 (clone 2GV6) is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. It is a research-use only product.
Automatically generated - may contain errors
Lab products found in correlation
Ventana benchmark ultra, by Roche (2 mentions)
Anti foxp3 clone 236 a e7, by Thermo Fisher Scientific (1 mentions)
Anti cd68 clone kp 1, by Roche (1 mentions)
Anti pd 1 clone nat105, by Abcam (1 mentions)
Anti cd31 ab28364, by Abcam (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Anti cd4 clone sp35, by Roche (1 mentions)
Opal 7 color automation ihc kit, by Akoya Biosciences (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Anti ki 67 clone 30 9, by Roche (1 mentions)
Anti cd20 clone l26, by Roche (1 mentions)
Prolong diamond antifade mounting, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti cd3 clone 2gv6
1
Quantifying Immune Cell Markers in FFPE Samples
2
Comprehensive Immunohistochemical Analyses
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Immunohistochemical analyses were performed using a standard protocol detailed previously.35 36 (link) Antigens were retrieved in 10 mM citrate (pH 6) (Sigma Aldrich) or EDTA (pH 9) (Zytomed Systems, Berlin, Germany) buffer. The primary antibodies used in the present study were as followed: anti-HMGB1 (clone 1D5, Abnova, Taipei City, Taiwan), anti-cleaved caspase 3 (clone C92-605, BD Biosciences), anti-CD31 (ab28364, Abcam, Cambridge, UK), anti-CD3 (clone 2GV6, Ventana Medical Systems, Tucson, Arizona, USA), anti-Foxp3 (clone 236 A/E7; eBioscience, San Diego, California, USA), anti-CD68 (clone KP-1, Ventana Medical Systems), anti-CD206 (clone E2L9N, Cell Signaling Technology, Danvers, MA, USA), anti-PD-1 (clone NAT105, Abcam), anti-human PD-L1 (clone 28–8, Abcam) and anti-mouse PD-L1 (orb10162, Biorbyt, Cambridge, UK). The secondary reaction was performed using the mouse or rabbit Envision+system (Dako, Glostrup, Denmark) according to the manufacturer’s recommendations. Positive cells were visualized using a 3,3’-diaminobenzidine (DAB) substrate (Cell Signaling Technology). Mouse or rabbit control IgGs (Santa Cruz Biotechnology, Santa Cruz, California, USA) were used as negative controls.
3
Immunophenotyping of FFPE Tumor Samples
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FFPE blocks for 207 patients with sequencing data were sectioned and stained for anti-CD3 (clone 2GV6, predilute; Ventana Medical Systems), anti-CD4 (clone SP35, predilute; Ventana Medical Systems), anti-CD8 (clone SP57, predilute; Ventana Medical Systems) and anti-PD-1 (clone SP263, predilute; Ventana Medical Systems) using an automated immunostainer (Ventana BenchMark ULTRA; Ventana Medical Systems, Tucson, AZ) without any dilution. Stained slides were digitised using an Aperio AT2 whole slide scanner. CD3, CD4 and CD8 staining was quantified using the Aperio Positive Pixel digital pathology tool and PDL-1 expression was determined using the Combined Positive Score system. The data were further verified by a pathologist (PR).
4
Multiplex Immunohistochemistry for Tissue Analysis
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Tissue sections were prepared from formalin-fixed paraffin embedded tissue blocks and cut to 4 μm serial sections and mounted on Superfrost Plus (VWR). The procedure for multiplex immunohistochemistry (mIHC) was followed by a manufacturer’s protocol for Opal7-color automation IHC kit (Akoya Bioscience), and the staining was performed with Autostainer DISCOVERY ULTRA (Ventana). Antibodies used in mIHC are anti-CD3 (clone 2GV6, Ventana), anti-CD20 (clone L26, Ventana), anti-Ki67 (clone 30-9, Ventana), anti-FOXP3 (clone SP97, Spring), anti-pan cytokeratin (CK; clone AE1/AE3, DAKO), anti-CD117 (clone c-kit, DAKO). The molecular markers of immune panel (CD3, CD20, Ki67, CKs, FOXP3, and CD117) were visualized with Opal520, Opal540, Opal570, Opal620, Opal650, and Opal690, respectively. DAPI counterstaining was performed with Discovery QD DAPI (Roche). ProLong Diamond Antifade Mounting (ThermoScientific) was used for mounting the coverslip. Detailed staining conditions and autostainer’s protocols are reported in our recent report86 (link).
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