Immunofluorescence double staining was performed to assess the number of CD4þ/CD3þ and CD8þ/CD3þ T cells. The frozen sections were fixed in acetone for 10 min and washed with TBST. Nonspecific endogenous peroxidase activity was blocked by exposure to 0.03% hydrogen peroxidase in 100% methanol for 5 min, and masking was conducted with 5% goat serum albumin in phosphate-buffered saline for 5 min at room temperature. Incubation was carried out overnight at 4% using an anti-CD3 antibody (diluted 1:800, Dako, Glostrup, Denmark) and an anti-CD4 antibody (diluted 1:100, Serotec, Kidlington, UK) or an anti-CD3 antibody (diluted 1:800, Dako) and an anti-CD8 antibody (diluted 1:200, Serotec). The slides were subsequently rinsed with TBST, treated for 60 min at room temperature with Alexa Fluor 488-or 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA), and rinsed with TBST. Then, nuclei were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI; Invitrogen). As a negative control, mouse or rabbit isotype immunoglobulin, diluted to the same concentration, was substituted for the primary antibody. The number of double-positive cells per 100 mm 2 was calculated.
Anti cd3 antibody
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The Anti-CD3 antibody is a laboratory reagent used in flow cytometry and other immunological applications. It binds to the CD3 complex on the surface of T cells, which is essential for T cell activation and function. The antibody can be used to identify and quantify T cell populations in biological samples.
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Lab products found in correlation
Axio scan z1, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Lamina multilabel slide scanner, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti claudin 1, by Abcam (1 mentions)
Anti pcna antibody, by Abcam (1 mentions)
Anti caspase 3, by Proteintech (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Anti cytokeratin 18, by Abcam (1 mentions)
Anti iga antibody, by Abcam (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Dulbecco s modified eagle s medium, by Corning (1 mentions)
Primary anti cd68 antibody, by Agilent Technologies (1 mentions)
In situ cell death detection kit, by Merck Group (1 mentions)
Anti cd68 antibody, by Bio-Rad (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Hitrap hp chelating columns, by GE Healthcare (1 mentions)
Bond polymer refine kit, by Leica (1 mentions)
Bond rx automated system, by Leica (1 mentions)
16 protocols using anti cd3 antibody
1
Immunofluorescence Quantification of T-Cell Subsets
2
Colonic Inflammation Assessment Protocol
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The presence of inflammatory changes was assessed as previously described by performing a neutrophil and lymphocyte count on 10 different colonic fields at 40 x magnification [12 (link)]. Hematoxylin-eosin staining was performed. Lymphocytes were identified with anti-CD3 antibodies (ready to use rabbit anti human polyclonal antibodies, DAKO, Copenhagen, Denmark), for neutrophils anti-CD15 antibodies (ready to use mouse anti human monoclonal antibodies, DAKO, Copenhagen, Denmark) were used. The number of lymphocytes and neutrophils were scored both at the bottom of the crypts and in the crypts as a whole. For histological evaluation the means of lymphocyte and neutrophils infiltrate were compared using the Mann-Whitney test. A P value of <0.05 was considered statistically significant.
3
Quantifying Lung Metastases and T-cell Infiltration
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Lungs were perfused with 5 ml of PBS and fixed in 10% neutral-buffered formalin in saline. Lungs were embedded into paraffin, and 5-μm sections were cut. To determine metastatic burden lung sections were stained with haematoxylin and eosin and then scanned using a Zeiss Axio Scan.Z1 to visualise the entire lung. To enumerate CD3+ T cell infiltration into the metastatic nodules, lungs were stained with anti-CD3 antibodies (Dako) prior to detection with DAB chromogen and counterstaining with haematoxylin. Slides were scanned and CD3+ T cells enumerated per metastatic nodule using Zen software (Zeiss).
4
Nerve and Skin Immunohistochemistry in Mice
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Mice (n = 5 per group) were perfused and sciatic nerves and hindfoot skin biopsies were dissected and prepared as previously described [38] . Sections were double stained by first incubating with rabbit polyclonal antibodies directed at the microglia/macrophage cytoplasmic calcium adaptor (anti-Iba-1) for the detection of macrophages (1:200; WAKO Chemicals USA, Richmond, VA) or with rabbit polyclonal anti-CD3 antibodies for detection of T-cells (1:200; Dako Cytomation, Hamburg, Germany). Second, the mouse monoclonal antibody against neurofilament 200 (NF200; 1:500; Sigma Aldrich, Taufkirchen, Germany) was used to identify nerve axons or the mouse monoclonal antibody against CD68 (ED1) for the detection of activated macrophages (1:200, Abcam, Cambridge, UK). For identification of intraepidermal small nerve fibers, polyclonal antibody against the axonal protein gene product (PGP 9.5; 1:1000; Abcam) was used. Next, the immunostaining was conducted as described elsewhere [38] .
5
Histological Analysis of Tumor-Infiltrating T Cells
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Histological Tumors were fixed in 4% neutral buffered formalin and embedded in paraffin. Sections (4μm) were stained with hematoxylin-eosin (H&E) and subjected to microscopic analysis. To investigate T cells infiltration, sections were stained with an anti-CD3 antibody (Dako, ref: A0452) or rabbit IgG isotype control. Histological analysis was performed at the HistIM facility of Cochin Institute (Paris, France). Slides were imaged using a Lamina multilabel slide scanner (Perkin Elmer). For quantitative analysis of T cells infiltration, six different and noncontiguous representative fields (40x magnification) were randomly selected for each experiment and their areas were quantified for immunoreactive CD3.
6
Porcine Reproductive Virus Research Protocol
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VERO (ATCC®CRL-1586) and PK-15 cells (ATCC®CCL-33) were cultured in Dulbecco’s modified Eagle’s medium (CORNING, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The PRV strain ZJ 01 was isolated from a pig herd at China in 2012 (Gu et al., 2015 (link)), which was used for all experiments. The PRV were proliferated in PK-15 cells and stored at −80°C. β-Streptococcus equinus strain SheepZ001 were cultured in Tryptone soybean broth (TSB) + 0.05% fetal bovine serum (FBS) medium at 37°C, 220 rpm. Pasteurella multocida HB01 (Serotype A) were cultured in Brain-heart extract broth (BHI) medium at 37°C, 220 rpm. Bacillus subtilis 168, WB800 and other Bacillus subtilis group isolated from sheep nasal cavity were cultured in Luria-Bertani (LB) medium at 37°C, 220 rpm. Anti-PRV gB-protein monoclonal antibodies (1B1, prepared and stored in our laboratory), anti-GAPDH antibody (Proteintech), anti-cytokeratin18 (Abcam), anti-claudin 1(Abcam), anti-caspase3 (Proteintech); anti-caspase9 (Proteintech); anti-ISG15 antibody (Abcam), anti-PCNA antibody (Abcam), anti-CD3 antibody (Dako), anti-IgA antibody (Abcam) and anti-CD208 antibody (Dendritics) was used in the study.
7
Immunohistochemical Analysis of Bone Marrow
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Thin sections of paraffin-embedded bone marrow were stained with hematoxylin and eosin. Activated macrophages were evaluated by immunohistochemical staining with primary anti-CD68 antibody (DAKO, Kyoto, Japan) and antibody against galactin-3 (Nichirei, Tokyo, Japan), a carbohydrate-binding lectin secreted by activated macrophages [14 (link)]. A rabbit polyclonal anti-CD3 antibody (DAKO) was used to determine the number of T lymphocytes. The Berlin blue stain was performed to detect intra- and extracellular iron deposition [8 (link)]. The sections were evaluated by two different pathologists who specialize in the pathophysiological analysis of infection and inflammatory diseases [15 (link), 16 (link)].
8
Abdominal Aortic Aneurysm Histological Analysis
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The suprarenal region of the abdominal aorta containing AAAs was serially cross-sectioned (8 μm sections). Paraffin embedded aortas were used for different stainings. Elastin staining was visualized using Orcein, the mean number of elastin layers was quantified for each mice. The extent of vascular smooth muscle cells (VSMC) was evaluated using anti-αSMC (Sigma-Aldrich) within media. T lymphocytes were detected using anti-CD3 antibody (Dako), macrophages using anti-CD68 antibody (AbD serotec). IHC chromogen substrate AEC (Thermo scientific) was used for revelation. The extent of apoptosis cells was evaluated by Terminal dUTP nick end-labelling (TUNEL) staining, using In Situ Cell Death Detection Kit (Sigma-Aldrich). We performed morphometric studies using Histolab software (Microvisions).
9
Quantification of Tumor-Infiltrating Lymphocytes
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After microscopic examination, an area of 2 mm in diameter was marked on the tumor center, and two areas were marked on the invasive front. Tissue blocks were available for 453 cases, and the marked areas of the corresponding tissue blocks were punched to construct a tissue microarray (TMA). Four-micrometer-thick sections from TMA tissue blocks were stained with an anti-CD3 antibody (1:200, Dako, Glostrup, Denmark) and anti-CD8 antibody (1:100, Neomarkers, Fremont, CA, USA). CD3-positive or CD8-positive cells were counted using the open-source software QuPath25 (link). The output was the CD3 TIL density and CD8 TIL density (number of cells per mm2 of tissue) of each core.
10
Antibody and Reagent Sources for Cell Signaling
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