Multiplex immunofluorescence staining and analysis was performed on 5-micron thick FFPE tissue sections as previously described using a Bond RX autostainer2 ,29 (link). Following deparaffinization, rehydration, and antigen retrieval, slides were serially stained with antibodies for SOX10 (EP268; Epitomics), CD8 (4B11, Leica), FOXP3 (D608R, Cell Signaling), PD-1 (EPR4877(2), Abcam), and PD-L1 (E1L3N, Cell signaling); each followed by anti-rabbit polymeric horseradish peroxidase (BOND Polymer Refine Detection Kit, Leica Biosystems) with labelling by Opal Fluorophore Reagents - as previously described. Stained slides were subsequently coverslipped with Prolong Diamond Anti-fade mounting medium (no. P36965, Life Technologies) and imaged using the Vectra multispectral imaging platform (PerkinElmer, Hopkinton, MA). Between 5 and 11 fields of view at 20x resolution were acquired and analyzed using supervised machine learning algorithms within the Inform 2.4 software (PerkinElmer), in order to assign spatial coordinates and cell phenotypes to all cells.
Cd8 4b11
Manufactured by Leica
Sourced in United States
The CD8 (4B11) is a laboratory equipment product designed for the detection and analysis of CD8+ T cells. It functions as a cell surface marker, allowing for the identification and quantification of CD8-positive cells in various biological samples. The core function of this product is to provide researchers and clinicians with a reliable tool for immunophenotyping and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 ps1, by Santa Cruz Biotechnology (2 mentions)
Cd4 368, by Leica (2 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Epr4877 2, by Abcam (1 mentions)
Inform 2, by PerkinElmer (1 mentions)
Vectra multispectral imaging platform, by PerkinElmer (1 mentions)
E1l3n, by Cell Signaling Technology (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
Ultraview universal dab detection kit, by Agilent Technologies (1 mentions)
Benchmark xt, by Roche (1 mentions)
Il 1β 3a6, by Cell Signaling Technology (1 mentions)
Foxp3 236a e7, by Abcam (1 mentions)
Opal 7 color ihc kit, by Akoya Biosciences (1 mentions)
Bond rx, by Leica (1 mentions)
6 protocols using cd8 4b11
1
Multiplex Immunofluorescence Profiling of FFPE Tissue
2
Immunohistochemical Analysis of Hepatic Cytolysis
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Three patients with persistent hepatic cytolysis underwent liver and digestive system biopsies.
In addition, one patient underwent skin biopsy twice for rashes. The biopsy samples were fixed in formalin and embedded in paraffin. Four-micrometer sections were generated and stained with hematoxylin-eosin-saffron. An immunohistochemical study was carried out using an automated immunohistochemistry system with antibodies against the following: CD20 (L26, Dako, 1/20e), CD3 (polyclonal rabbit, Dako, 1/100e), CD4 (4B12, Leica, 1/40e), CD8 (4B11, Leica, 1/50e), NLRP3 (HPA012878, Atlas antibodies, 1/400e), IL-1β (3A6, Cell Signaling, 1/100e), and NF-κB p65 (D14E12, Cell Signaling, 1/1500e). Briefly, 4-µm sections were prepared from paraffin blocks and placed on Superfrost plus slides. After antigen unmasking with target retrieval solution, citrate pH 6 or 9 was added according to the antibodies to be used, and the immunohistochemical study was carried out by an immunohistochemistry automaton (Ventana Benchmark XT). After incubation with the primary antibody (20 to 60 min), detection was carried out with an Ultraview Universal DAB Detection Kit (Dako) indirect detection kit according to the supplier's instructions.
A biopsy sample of chronic active colitis was used as an immunohistochemical control for some antibodies.
In addition, one patient underwent skin biopsy twice for rashes. The biopsy samples were fixed in formalin and embedded in paraffin. Four-micrometer sections were generated and stained with hematoxylin-eosin-saffron. An immunohistochemical study was carried out using an automated immunohistochemistry system with antibodies against the following: CD20 (L26, Dako, 1/20e), CD3 (polyclonal rabbit, Dako, 1/100e), CD4 (4B12, Leica, 1/40e), CD8 (4B11, Leica, 1/50e), NLRP3 (HPA012878, Atlas antibodies, 1/400e), IL-1β (3A6, Cell Signaling, 1/100e), and NF-κB p65 (D14E12, Cell Signaling, 1/1500e). Briefly, 4-µm sections were prepared from paraffin blocks and placed on Superfrost plus slides. After antigen unmasking with target retrieval solution, citrate pH 6 or 9 was added according to the antibodies to be used, and the immunohistochemical study was carried out by an immunohistochemistry automaton (Ventana Benchmark XT). After incubation with the primary antibody (20 to 60 min), detection was carried out with an Ultraview Universal DAB Detection Kit (Dako) indirect detection kit according to the supplier's instructions.
A biopsy sample of chronic active colitis was used as an immunohistochemical control for some antibodies.
3
Immunohistochemistry of FFPE Tissues
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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections using the avidin–biotin complex method as described previously [16 (link)]. We used 4-μm-thick serial sections of each FFPE blocks with antibodies against the following: CD3 (PS1; 1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD4 (368; 1:100), and CD8 (4B11; 1:200) from Leica Microsystems (Newcastle-upon-Tyne, UK), and FOXP3 (42; 1:100) produced in-house [4 (link)]. Immunohistochemistry without the primary antibody was performed as a negative control.
4
Immunohistochemical Analysis of Immune Markers
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Immunohistochemistry was carried out on formalin‐fixed, paraffin‐embedded tissue sections using the avidin–biotin complex method as described previously.41 We used 4‐μm‐thick serial sections of representative blocks with antibodies against the following: CD3 (PS1; 1:100) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD4 (368; 1:100) and CD8 (4B11; 1:200) from Leica Microsystems (Newcastle‐upon‐Tyne, UK), FOXP3 (42; 1:100) produced in house,18 BTLA (HPA047211; 1:500) from Atlas Antibodies (Stockholm, Sweden), and Cbl‐b (246C5A; 1:50) from Abcam (Cambridge, UK). Immunohistochemistry without the primary antibody was used as a negative control.
5
Tumor Immunophenotyping by IHC
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Immunohistochemical findings of formalin‐fixed, paraffin‐embedded tumor specimens taken at initial diagnosis, including PD‐L1 expression on cancer cells, CD8+ T cells, PD‐1+ immune cells, and FOXP3+ regulatory T cells, were examined. The antibody clones used were as follows: PD‐L1 (22C3, Dako, Santa Clara, CA, USA); CD8 (4B11, Leica Biosystems, Nussloch, Germany); PD‐1 (SP269, Spring Bioscience, Pleasanton, CA, USA); and FOXP3 (236A/E7, Abcam, Cambridge, UK). Positivity of PD‐L1 for tumor cells and of CD8, PD‐1, and FOXP3 for tumor‐infiltrating immune cells was expressed as a percentage of positive cells in the four representative areas. Additionally, the number of PD‐1+ immune cells was expressed as the density of positive cells in the four representative areas. Two independent experts blinded to the clinical data evaluated the immunohistochemical findings. If the assessments differed, the experts re‐evaluated the specimen and reached a consensus.
6
Multiplex Immunohistochemistry Panel for Tumor Microenvironment
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Paraffin sections (4 μm) were sequentially stained for six markers plus DAPI using the Opal 7-color IHC kit (Akoya Biosciences, Marlborough, MA). Automated dewaxing, antigen retrieval, and staining were performed on a BondRX autostainer (Leica Biosystems, Lincolnshire, IL). The antibodies used recognized the same antigens detected in frozen sections wherever possible. The CD68 marker was replaced with CD163 for this analysis. Markers used primary antibodies against CD3 (LN10; Leica Biosystems), CD4 (N1UG0, Invitrogen), CD8 (4B11, Leica Biosystems), CD163 (10D6, Leica Biosystems), CD1a (O10; Santa Cruz Biotechnology), and Ki-67 (B56; BD Biosciences) along with the Opal dyes and DAPI (BD Biosciences) were titrated to determine optimal concentrations, pairing each primary antibody with an Opal dye and defining the preferred staining order. The spectral library created for these stainings serves as the control for multispectral imaging and allows for the subtraction of any background fluorescence.
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