Formalin-fixed, paraffin-embedded labial and parotid salivary gland tissue samples were serially sectioned at 3 µm thickness and deparaffinized. Automatic staining was performed with H&E, and tissue samples were manually stained for CD3 (clone 2GV6, Roche), high-molecular-weight cytokeratin (hmwCK, clone 34βE12, Roche) and CD20 (clone L-26, Roche). Antigen retrieval was performed for 15 min in EDTA buffer (98°C, pH 8.0), and endogenous peroxidase was blocked. Pre-fixed dilutions of primary antibodies (1% BSA-PBS, Roche) were applied for 75 min. Primary antibodies were visualized by using 3,3′-diaminobenzidine (DAB) after incubation with a poly-horseradish peroxidase-labelled secondary antibody (Thermo Scientific). Tonsillar tissue was used as both a positive and negative control, as the tonsillar epithelium expresses hmwCK and does not express CD3 or CD20, and the tonsillar lymphoid tissue expresses CD3 and CD20 but does not express hmwCK.
Cd3 clone 2gv6
Manufactured by Roche
Sourced in United States
The CD3 (clone 2GV6) is a laboratory reagent used for the identification and enumeration of T cells. It is a monoclonal antibody that binds to the CD3 complex, a group of membrane-bound proteins associated with the T cell receptor. This reagent can be used in flow cytometry and other immunoassays to detect and quantify T cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cd20 clone l26, by Agilent Technologies (5 mentions)
Cd4 clone sp35, by Roche (4 mentions)
Cd8 clone sp57, by Roche (3 mentions)
Cd20 clone l26, by Roche (2 mentions)
Cd68 clone kp 1, by Roche (2 mentions)
Cd163 clone mrq 26, by Roche (2 mentions)
Benchmark ultra, by Roche (2 mentions)
Pd l1 clone e1l3n, by Cell Signaling Technology (2 mentions)
Cd68 clone pg m1, by Agilent Technologies (2 mentions)
Tia 1 clone tia1, by Biocare Medical (2 mentions)
Bcl2 clone 124, by Agilent Technologies (2 mentions)
Cd10 clone 56c6, by Leica (2 mentions)
Bond max autostainer, by Leica (2 mentions)
Myc clone ep121, by Cell Marque (2 mentions)
Bcl6 clone ln22, by Leica (2 mentions)
Mum1 clone ma695, by Leica (2 mentions)
Ventana benchmark xt, by Roche (2 mentions)
Cd8 clone 144b, by Agilent Technologies (2 mentions)
Bsa pbs, by Roche (1 mentions)
Cd8 clone c8 144b, by Agilent Technologies (1 mentions)
11 protocols using cd3 clone 2gv6
1
Comprehensive Immunohistochemical Characterization of Salivary Gland Tissues
2
Comprehensive Immunophenotyping of Tissue Samples
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We performed immunohistochemical stains on formalin-fixed paraffin-embedded three micron tissue sections using an automated immunostainer Benchmark Ultra (Roche) with an Ultra-View Universal DAB brown detection kit (alongside an Ultra-View Universal AP Red detection kit for double staining). These antibodies were used for the following targets: CD3 (clone 2GV6, predilute Roche Cat # 790-4341), CD4 (clone SP35, predilute Roche Cat# 790-4423), CD8 (clone SP57, predilute Roche Cat#790-4460), granzyme B (clone GrB-7+D170, dilution 1:100, Millipore Cat# MAB 3070), perforin (clone KM585 PI-8) (dilution 1:200, Leica Cat# NCL-L-perforin) detection with Optiview kit (Roche), CD68 (clone KP1, predilute Roche Cat#790-2931), CD163 (clone MRQ-26, predilute Roche Cat#760-4437).
3
Monoclonal Antibody Characterization for IHC
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Monoclonal antibody (mAb) HCA2, which recognizes β2m-free HLA-A (excluding -A24), -B7301 and -G heavy chains [23 (link)], and mAb HC10, which recognizes β2m-free HLA-A3, -A10, -A28, -A29, -A30, -A31, -A32, -A33 and all β2m-free -HLA-B (excluding -B5702, -B5804 and -B73) and -HLA-C heavy chains [24 (link), 25 (link)], were developed and characterized as previously described. mAbs were purified from ascitic fluid by affinity chromatography on Protein G columns. The purity and specific reactivity of mAb preparations were assessed by SDS-PAGE, binding assays and western blotting, respectively. A pool of mAb HCA2 and HC10 (ratio: 1:1) was used for immunohistochemistry (IHC) of tissues sections.
The IFNAR1- (ab62693, Abcam), CD3- (clone 2GV6, Ventana, Roche), CD8- (clone C8/144b, Dako), and PD-L1- (clone 22C3 pharmDx, Dako) specific mAbs were purchased from the indicated companies.
The IFNAR1- (ab62693, Abcam), CD3- (clone 2GV6, Ventana, Roche), CD8- (clone C8/144b, Dako), and PD-L1- (clone 22C3 pharmDx, Dako) specific mAbs were purchased from the indicated companies.
4
Comprehensive Immunohistochemical Profiling
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Immunohistochemistry (IHC) was performed on 4 µm thin FFPE tissue sections as previously described [29 (link)], using automated slide stainers BenchMark (Ventana Medical System-Roche) and Leica Bond-III, (Leica Biosystems,), according to manufacturer’s instructions. To characterize lymphocytic infiltrate, CD3 (clone 2GV6, ready to use), CD4 (clone SP35, ready to use), and CD8 (clone SP57, ready to use) monoclonal antibodies (Roche) were applied on tissue sections for 15 min at 25 °C. Monoclonal antibody directed to nuclear transcription factor forkhead box P3 (FOXP3) (clone D2W8E, 1:250, Cell Signaling Technology) was utilized to identify regulatory T cells. CD68 (clone KP-1, ready to use, Roche), and CD163 (clone MRQ-26, Roche) monoclonal antibodies were employed to identify macrophages and M-2 like macrophages, respectively [33 (link)]. Intra-tumoral vascularization was assessed by using CD31 monoclonal antibody (clone JC70A, ready to use, Dako). Sections were counterstained with hematoxylin.
5
Quantitative Renal Immune Cell Profiling
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Three μm-thick sections were cut from 1 core formalin-fixed and paraffin-embedded tissue of a 16-gauge needle biopsy and stained using antibodies against CD3 (clone 2GV6, Roche), CD4 (clone SP35, Roche), CD8 (clone SP57, Roche), CD20 (clone L26, Roche), CD68 (clone PGM1, Diagnostic Biosystems), CD56 (clone MRQ42, Roche), CD138 (clone B-A38, Roche) and TIA1 (2G9A10F5, Vitro Máster Diagnóstica). Here, TIA1 marker was used as a surrogate marker of cytotoxic T and NK cell granules. 39 39. Tian, Q. Immunostains were automatically performed with a Benchmark XT (Roche) and revealed with DAB Optiview Kit (clone B-A38, Roche). Stained inflammatory cells were quantitatively assessed by 2 pathologists blinded to clinical background under light microscopy in the glomerular capillaries, peritubular capillaries, and interstitial compartment, especially when forming aggregates in scarred areas. A standardized evaluation of the cell count in renal parenchyma was conducted according to renal compartments. In glomerular capillaries, cell count was standardized by dividing the number of cells by the total glomeruli. In peritubular capillaries and renal interstitial compartments, cell count was standardized by dividing the number of cells by the length of the sample in millimeters.
6
Immunohistochemical Profiling of Ovarian Carcinoma
7
Immunohistochemical Profiling of Lymphoid Tissues
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Tissue sections were subjected to the same deparaffinization, antigen retrieval, and blocking protocol mentioned above. The lymph node and spleen sections were stained with primary antibodies for CD3 (clone 2GV6, Ventana), CD4 (Clone SP35, Ventana), CD8 (clone 144B, Agilent), CD20 (Clone L26, Agilent), and TIA-1 (clone TIA1, Biocare).
8
Immunohistochemical and FISH Analysis of DLBCL
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IHC was prospectively performed on the routine diagnostic basis using representative whole formalin-fixed paraffin-embedded (FFPE) tissue sections and antibodies against CD3 (clone 2GV6; Ventana Medical Systems, Tucson, AZ, USA), CD20 (clone L26; DAKO, Carpinteria, CA, USA), BCL2 (clone 124; DAKO, Carpinteria, CA, USA), BCL6 (clone LN22; Novocastra, Newcastle, UK), CD10 (clone 56C6; Novocastra), MUM1 (clone Ma695; Novocastra), MYC (clone EP121; Cell Marque, Rocklin, CA, USA), and Ki-67 (clone MIB-1; DAKO). Staining was performed using a Ventana Benchmark XT (Ventana Medical Systems) or a Bond-Max autostainer (Leica Microsystems, Melbourne, Australia). COO was determined using the IHC-based Hans algorithm as previously described [29 (link)]. DE status was defined as the co-expression of MYC (in ≥40% of tumor cells) and BCL2 (in ≥70% of tumor cells) as previously described [12 (link)]. TMA was constructed using the FFPE tissue blocks from 50 selected cases with DLBCL, and we performed MYC, BCL2, BCL6 FISH on the TMA. FISH was performed using Vysis LSI BCL2 Dual Color Break Apart Rearrangement Probe (Vysis, Downers Grove, IL, USA), Vysis LSI BCL6 Dual Color Break Apart Rearrangement Probe (Vysis), and Vysis LSI MYC Dual Color Break Apart Rearrangement Probe (Vysis)).
9
Immunohistochemical Profiling of B-cell Lymphomas
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IHC was performed using antibodies against CD3 (clone 2GV6; Ventana Medical Systems, Tucson, AZ), CD20 (clone L26; DAKO, Carpinteria, CA), BCL2 (clone 124; DAKO), BCL6 (clone LN22; Novocastra, Newcastle, UK), CD10 (clone 56C6; Novocastra), MUM1 (clone Ma695; Novocastra), MYC (clone EP121; Cell Marque, Rocklin, CA), Ki-67 (MIB-1; Ventana Medical Systems), PD-1 (clone MRQ-22; Cell Marque), IgD (clone DRN1C; Novocastra), and FOXP1 (clone SP133; Cell Marque) on representative whole FFPE tissue sections. Epstein-Barr virus in situ hybridization was performed using the Bond Ready-to-Use ISH EBER probe (Leica Biosystems, Newcastle, UK) or the INFORM EBER probe (Ventana Medical Systems). Immunostaining was performed using Ventana Benchmark XT (Ventana Medical Systems) or Bond-Max autostainer (Leica Microsystems, Melbourne, Australia) according to the manufacturer’s protocol.
B-cell monoclonality was detected using the IdentiClone IGH Gene Clonality Assay (Invivoscribe Technologies Inc., San Diego, CA).
B-cell monoclonality was detected using the IdentiClone IGH Gene Clonality Assay (Invivoscribe Technologies Inc., San Diego, CA).
10
Automated Multiplex Immunohistochemistry for Tissue Analysis
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Immunohistochemical staining was performed on 2 µm paraffin sections with an automated IHC staining system (Ventana BenchMark ULTRA, Ventana Medical Systems, Italy). Sequential double IHC was performed on Ventana BenchMark ULTRA, using a ultraView Universal DAB detection Kit as the first stain and ultraView Universal Alkaline phosphatase Red detection kit as the second stain. Heat-induced epitope retrieval pre-treatment was performed using CC1 buffer (standard CC1, Roche Ventana) by boiling for 36 min for both CD31 and CD3 and for 64 min for RORγt. Afterwards, slides were incubated with primary antibodies: CD31 antibody (clone JC70, Cell Marque, dilution 1:20) for 16 min at 37°C or CD3 (clone 2GV6, Ventana, dilution 1:20) for 44 min at 37°C and RORγt (clone 6F3.1, Millipore, dilution 1:20) for 36 min at 37°C. CD31 and CD3 were visualized with DAB chromogen, and RORγt was visualized with Fast Red chromogen.
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