Immunohistochemistry (IHC) for the detection of CD68+ and CD3+ cells in the hernia was performed by the NovolinkTM Polymer Detection Kit (Leica Biosystems, Newcastle, UK) following the manufacturer’s instructions. Antigen retrieval was performed through incubation in near-boiling point 10 mmol/L sodium citrate buffer with a pH of 6.0 for 1 min, followed by incubation with 20 μg/mL proteinase K (Sigma-Aldrich Inc., St Louis, MO, USA) solution for 15 min at 37 °C. Sections were incubated with anti-CD68 (clone ED1, 1:100 dilution, Bio-Rad Laboratories, Irvine, CA, USA) or anti-CD3 (undiluted, Leica Biosystems, Newcastle, UK) antibodies overnight at 4 °C. The stained sections were imaged with light microscopy. CD3+ and CD68+ cells were quantified using ImageJ tools directly on the acquired images. From these, the %CD3 positivity was normalized to the total number of cells in the hernia area, and the %CD68 positivity was normalized to the area of the hernia.
Anti cd3
Manufactured by Leica
Sourced in United Kingdom
Anti-CD3 is a monoclonal antibody that specifically binds to the CD3 complex on the surface of T cells. The CD3 complex is essential for T cell activation and signal transduction. Anti-CD3 can be used in a variety of laboratory applications to study T cell function and activation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd68 clone ed1, by Bio-Rad (1 mentions)
Novolink polymer detection kit, by Leica (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Anti cd4, by Leica (1 mentions)
Anti cd8, by Leica (1 mentions)
Histovt one, by Nacalai Tesque (1 mentions)
Anti cd68, by Agilent Technologies (1 mentions)
2 4 amidinophenyl 1h indole 6 carboxamidine dapi, by Vector Laboratories (1 mentions)
Tcs sp8 x, by Leica (1 mentions)
Mca771g, by Bio-Rad (1 mentions)
Histoquest software, by TissueGnostics (1 mentions)
Osteomeasure image analysis system, by OsteoMetrics (1 mentions)
Anti il 8, by Abcam (1 mentions)
Anti ll37, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti cd3
1
Quantifying CD68+ and CD3+ Cells in Hernia Tissue
2
SARS-CoV-2 Lung Histopathology and Immune Profiling
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Between August 2020 and March 2021, histological lung specimens were obtained from eight SARS-CoV-2-positive patients for antemortem evaluation after bronchoscopy and cryotransbronchial biopsy. The clinical records, radiological, and pathological findings were retrospectively compiled. Transbronchial biopsy specimens were available in all patients, taken from the most prominent areas of the interstitial ground-glass opacities. The mean number of lung biopsy fragments per case was three (range 2–4).
Biopsy specimens were fixed in 10% formalin, embedded in paraffin wax, and sectioned at 5 μm. Sections were stained with haematoxylin and eosin. Each biopsy specimen was evaluated semi-quantitatively for the following parameters: the presence or absence of reactive pneumocytes; alveolar macrophages; lymphocytes; neutrophils; plasma cells, fibroblastic foci; peribronchiolar inflammation; and viral cytopathic effect.
Immunohistochemical labelling was performed with antibodies directed against the following proteins: CD3 (anti-CD3; Leica Biosystems, UK); CD4 (anti-CD4; Leica Biosystems); CD8 (anti-CD8; Leica Biosystems); CD20 (anti-CD20; Leica Biosystems); and SARS-CoV-2 nucleocapsid protein.2 (link) CD4 and CD8 staining were scored as percent-positive immune cell: 0, negative; 1, 1–30%; 2, 31–70%; and 3, 71–100% positive immune cell.
Biopsy specimens were fixed in 10% formalin, embedded in paraffin wax, and sectioned at 5 μm. Sections were stained with haematoxylin and eosin. Each biopsy specimen was evaluated semi-quantitatively for the following parameters: the presence or absence of reactive pneumocytes; alveolar macrophages; lymphocytes; neutrophils; plasma cells, fibroblastic foci; peribronchiolar inflammation; and viral cytopathic effect.
Immunohistochemical labelling was performed with antibodies directed against the following proteins: CD3 (anti-CD3; Leica Biosystems, UK); CD4 (anti-CD4; Leica Biosystems); CD8 (anti-CD8; Leica Biosystems); CD20 (anti-CD20; Leica Biosystems); and SARS-CoV-2 nucleocapsid protein.2 (link) CD4 and CD8 staining were scored as percent-positive immune cell: 0, negative; 1, 1–30%; 2, 31–70%; and 3, 71–100% positive immune cell.
3
Immunofluorescent Staining of IL-38 and Cell Markers
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The formalin-fixed and paraffin-embedded sections were deparaffinized and incubated with Histo VT One (Nacalai Tesque, Kyoto, Japan) for antigen retrieval according to the manufacturer’s instructions. After cooling, an anti-IL-38 antibody (Abcam, Cambridge, UK) was applied and incubated overnight at 4°C. DyLight 549-labeled anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) was used as a secondary antibody. For double-staining procedures, anti-IL-38, anti-CD3 (Leica Biosystems, Buffalo Grove, IL), anti-CD19 (Santa Cruz Biotechnology, Dallas, TX), anti-CD68 (DAKO, Santa Clara, CA), and anti-myeloperoxidase (MPO) antibodies (R&D systems, Minneapolis, MN) were applied and incubated overnight at 4°C. Then, the slides were incubated with secondary antibodies conjugated to fluorescent dyes for 1 h at room temperature, visualizing the nuclei with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (Vector Laboratories). Images were obtained with a confocal microscope TCS SP8 X (Leica Microsystems, Wetzlar, Germany). The antibodies used are listed in Supplemental Table 1* .
4
Histopathological Analysis of Murine Joints
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Hind paws were prepared and analyzed by using previously described histopathologic techniques [32 (link)-35 (link)]. Staining with H&E allowed a general assessment, and toluidine blue (TB) destaining was performed to determine cartilage matrix loss. Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify osteoclasts. Histomorphometric parameters (area of cartilage destruction, inflammation and erosion, as well as osteoclast numbers) were quantified by using the OsteoMeasure™ image analysis system (OsteoMetrics, Decatur, GA, USA).
Additional immunohistochemistry was done for T cells (anti-CD3; Novocastra Laboratories, Newcastle upon Tyne, UK), B cells (anti-CD45 receptor; BD Biosciences PharMingen, San Diego, CA, USA), macrophages (clone F4/80; AbD Serotec, Puchheim, Germany) and granulocytes (MCA771G; AbD Serotec) as reported previously [33 (link)-35 (link)], followed by quantitative analysis of the inflammatory infiltrate by tissue cytometry using HistoQuest™ software (TissueGnostics, Vienna, Austria) [36 (link),37 (link)].
Additional immunohistochemistry was done for T cells (anti-CD3; Novocastra Laboratories, Newcastle upon Tyne, UK), B cells (anti-CD45 receptor; BD Biosciences PharMingen, San Diego, CA, USA), macrophages (clone F4/80; AbD Serotec, Puchheim, Germany) and granulocytes (MCA771G; AbD Serotec) as reported previously [33 (link)-35 (link)], followed by quantitative analysis of the inflammatory infiltrate by tissue cytometry using HistoQuest™ software (TissueGnostics, Vienna, Austria) [36 (link),37 (link)].
5
Immunohistochemistry of Pustular Skin Lesions
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For immunohistochemistry, paraffin embedded 4 µm thick tissue was used to the avidin-biotin complex method which was visualized by aminoethyl carbazole (GBI Labs, Bothell, WA, USA). Antigen retrieval was carried with pressure cooker of Cuisinart CPC-600 (Cuisinart Corp., East Windsor, NJ, USA) for 10 minutes in citrate buffer pH 6.0. All punch biopsy specimens from pustular skin lesions were stained with seven antibodies: anti-α-3-nAChR (Sigma, St. Louis, MO, USA; at 1:1,000) for the acrosyringia, anti-IL-23 for plaque psoriasis (BioLegend, San Diego, CA, USA; at 1:400), anti-IL-36R for pustular psoriasis (Abcam, Cambridge, UK; at 1:200), anti-LL37 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; at 1:200) for antimicrobial peptides, anti-IL-8 (Abcam; at 1:2,000), anti-Lipocalin-2 (LCN2) (Abcam; at 1:8,000) for neutrophils, and anti-CD3 for lymphocytes (Novocastra, Newcastle upon Tyne, UK; at 1:300). All slides were independently examined by two dermatologists for evaluating stain degree.
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