Anti-CD3 epsilon antibody [SP7]

26 product images

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  Flow Cytometry (Intracellular) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  Human peripheral blood lymphocytes stained with ab16669 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab16669, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

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  Image from Moussion C et al., PLoS One. 2008 Oct 6;3(10):e3331. Fig 2.; doi:10.1371/journal.pone.0003331; October 6, 2008, PLoS ONE 3(10): e3331.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  Immunohistochemical analysis of Human tonsil tissue, staining CD3 epsilon (green) with ab16669.
  
  Antigen retrieval was performed by heat mediation in citrate buffer (pH 6) and blocked with 5% goat serum and 5% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/100) overnight at 4°C. A Cy3®-conjugated anti-rabbit IgG was used as the secondary antibody.

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  Flow Cytometry (Intracellular) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon flow cytometry staining of Jurkat cells using rabbit anti-CD3 epsilon antibody
  
  

  This image was generated using a previous batch manufactured using hybridoma production method.

  Flow cytometric analysis of rabbit anti-CD3 epsilon (SP7) antibody ab16669 (1/100 dilution) in Jurkats cells (green) compare to negative control of rabbit IgG (blue).

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human tonsil using rabbit anti-CD3 epsilon antibody
  
  Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

  Merged staining of anti-PD1 (Anti-PD1 antibody [CAL20] ab237728; orange; Opal™520) anti-PDL1 (Anti-PD-L1 antibody [CAL10] ab237726; green; Opal™540) anti-CD68 (Anti-CD68 antibody [SP251] ab192847; yellow; Opal™570) anti-CD3 epsilon (ab16669; red; Opal™620) anti-Ki67 (Anti-Ki67 antibody [SP6] ab16667; light blue; Opal™650) and anti-PanCK (Anti-pan Cytokeratin antibody [C-11] ab7753; grey; Opal™690).

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT Akoya Biosciences^®).

  The section was incubated in six rounds of staining; in the order of Anti-PD1 antibody [CAL20] ab237728 (1/500 dilution), Anti-PD-L1 antibody [CAL10] ab237726 (1/500 dilution), Anti-CD68 antibody [SP251] ab192847 (1/300 dilution), ab16669 (1/300 dilution), Anti-Ki67 antibody [SP6] ab16667 (1/200 dilution) and Anti-pan Cytokeratin antibody [C-11] ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

  Sodium citrate antigen retrieval (Leica ER1 pH6.0 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round to avoid any cross-reactivity.

  DAPI (dark blue) was used as a nuclear counter stain.

  Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

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  This image is courtesy of a customer review submitted by Ying Li.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  ab16669 staining CD3 epsilon in Mouse Epidydimal fat pad tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue sections were fixed with formaldehyde, blocked with 5% serum for 4 hours at 25°C and permeabilized with Triton X-100. Samples were incubated with primary antibody (1/100 in PBST with BSA and goat serum) for 4°C at 12 hours. An Alexa Fluor® 568 goat anti-rabbit IgG (H + L) cross adsorbed was used as the secondary antibody.

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  Western blot - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  Lanes 1 - 6: Merged signal (red and green). Green – ab16669 observed at 23 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16669 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

  All lanes: Western blot - Anti-CD3 epsilon antibody [SP7] (ab16669) at 1/25 dilution

  Lane 1: THP1 whole cell lysate (-ve control) at 15 µg

  Lane 2: Raji whole cell lysate (-ve control) at 15 µg

  Lane 3: Jurkat whole cell lysate at 15 µg

  Lane 4: Human Thymus tissue lysate at 15 µg

  Lane 5: Mouse Thymus tissue lysate at 15 µg

  Lane 6: Rat Thymus tissue lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Performed under reducing conditions.

  Predicted band size: 19 kDa

  Observed band size: 23 kDa

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  This image is courtesy of a customer review submitted by Carl Hobbs

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  Immunohistochemical analysis of Formaldehyde fixed, paraffin-embedded rat spleen tissue sections labelling CD3 epsilon with ab16669 at a dilution of 1/100. Biotin conjugated Goat Anti-Rabbit IgG at 1/300 dilution was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid.

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  This image is courtesy of a customer review submitted by Dr Mal Niladri

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This image was generated using a previous batch manufactured using hybridoma production method.

  ab16669 staining rat infarcted heart tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

  Myocardial infarction was produced in a rat model following the ligation of the left anterior descending (LAD) coronary artery. Tissue was harvested 6 w following infarct, fixed with Histochoice for 72 hr, paraffin sectioned and the slide was then baked prior to CD3 epsilon staining. ab16669 at 1/200 was incubated overnight at 4°C. The image was taken with a confocal laser scanning microscope and shows cells giving strong inmmunofluorescence staining for CD3 epsilon antigen (green), indicating presence of cells of T-lymphocytes origin in the infarct zone of the heart tissue, counterstained nuclei with DAPI (blue). Note, CD3 epsilon tended to be present in nests of 2-5 cells that were non-uniformly distributed in the infarct zone. In addition, the image shows that the CD3 epsilon localization is predominantly membrane based and to a certain extent intracytoplasmic.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of mouse spleen using rabbit anti-CD3 epsilon antibody
  
  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen tissue labeling Sialoadhesin/CD169, CD3 epsilon and CD20 with Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and Anti-CD20 antibody [SP32] - BSA and Azide free ab236434 at 1:5000 dilution.
  
  Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD20 (gray; Opal™570) on mouse spleen.
  Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
  Panel C: anti-CD3 epsilon stained on T cells.
  Panel D: anti-CD20 stained on B cells.
  
  The section was incubated in three rounds of staining: in the order of Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840, ab16669, and Anti-CD20 antibody [SP32] - BSA and Azide free ab236434 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human colon using rabbit anti-CD3 epsilon antibody
  
  

  Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401 gray; Opal™690), anti-CD3 epsilon (ab16669 green; Opal™520) and anti-CD68 (Anti-CD68 antibody [EPR20545] ab213363 red; Opal™570) on human colon. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 epsilon stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401 (1/8000 dilution), ab16669 (1/150 dilution) and Anti-CD68 antibody [EPR20545] ab213363 (1/500 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0 epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human duodenum using rabbit anti-CD3 epsilon antibody
  
  

  Fluorescence multiplex immunohistochemical analysis of the human duodenum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401 gray; Opal™690), anti-CD3 epsilon (ab16669 green; Opal™520) and anti-CD68 (Anti-CD68 antibody [EPR20545] ab213363 red; Opal™570) on human duodenum. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-CD3 epsilon stained on T cells. Panel D: anti-CD68 stained on macrophages. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401 (1/8000 dilution), ab16669 (1/150 dilution) and Anti-CD68 antibody [EPR20545] ab213363 (1/500 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0 epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD19 (green; Opal™520), anti-CD19 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on human tonsil.
  Panel B: anti-CD19 staining the B lymphocytes in human tonsil.
  Panel C: anti-CD19 staining the B lymphocytes in human tonsil.
  Panel D: anti-CD3 staining the T lymphocytes in human tonsil.
  Nuclear DNA was labeled with DAPI (shown in blue).
  The section was incubated in three rounds of staining: in the order of Anti-CD19 antibody [EPR28949-559] ab320735 at 1/2000 dilution, Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution and ab16669 at 1/150 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The secondary used was Opal Polymer HRP Ms + Rb. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD19 (green; Opal™520), anti-CD19 (magenta; Opal™570) and anti-CD3 (yellow; Opal™690) on human Hodgkin's lymphoma.
  Panel B: anti-CD19 staining the B lymphocytes in human Hodgkin's lymphoma.
  Panel C: anti-CD19 staining the B lymphocytes in human Hodgkin's lymphoma.
  Panel D: anti-CD3 staining the T lymphocytes in human Hodgkin's lymphoma.
  Nuclear DNA was labeled with DAPI (shown in blue).
  The section was incubated in three rounds of staining: in the order of Anti-CD19 antibody [EPR28949-559] ab320735 at 1/2000 dilution, Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution and ab16669 at 1/150 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The secondary used was Opal Polymer HRP Ms + Rb. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD19 (green; Opal™520), anti-CD19 (green; Opal™570) and anti-CD3 (yellow; Opal™690) on human cerebrum.
  Panel B: anti-CD19 showed no staining in human cerebrum.
  Panel C: anti-CD19 showed no staining in human cerebrum.
  Panel D: anti-CD3 showed no staining in human cerebrum.
  Nuclear DNA was labeled with DAPI (shown in blue).
  The section was incubated in three rounds of staining: in the order of Anti-CD19 antibody [EPR28949-559] ab320735 at 1/2000 dilution, Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution and ab16669 at 1/150 dilution for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The secondary used was Opal Polymer HRP Ms + Rb. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD68 (gray; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, and counterstaining was with DAPI.
  
  Panel B: anti-CD3 epsilon stained on T cells with ab16669 at 1/500 dilution
  Panel C: anti-CD19 stained on B cells with Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution
  Panel D: anti-CD68 stained on macrophages with Anti-CD68 antibody [EPR20545] ab213363 1/500 dilution
  
  The section was incubated in three rounds of staining: in the order of Anti-CD68 antibody [EPR20545] ab213363 and ab16669 for 30 mins, then Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, and couterstaining was with DAPI.
  Panel B: anti-CD3 epsilon stained on T cells with ab16669 at 1/500 dilution
  Panel C: anti-CD19 stained on B cells with Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution
  Panel D: anti-CD68 stained on macrophages with Anti-CD68 antibody [EPR20545] ab213363 1/500 dilution
  The section was incubated in three rounds of staining: in the order of Anti-CD68 antibody [EPR20545] ab213363 and ab16669 for 30 mins, then Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human tonsil. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
  Panel B: anti-CD3 epsilon stained on T cells with ab16669 at 1/500 dilution
  Panel C: anti-CD19 stained on B cells with Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution
  Panel D: anti-CD31 stained on endothelial cells and immune cell subsets with Anti-CD31 antibody [EPR3094] - BSA and Azide free ab207090 at 1/500 dilution
  
  The section was incubated in three rounds of staining: in the order of Anti-CD31 antibody [EPR3094] - BSA and Azide free ab207090 and ab16669 for 30 mins, then Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  This data was developed using Anti-CD3 epsilon antibody [SP7] - BSA and Azide free ab205228, the same antibody clone in a different buffer formulation.

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD3 with Anti-CD3 epsilon antibody [SP7] - BSA and Azide free ab205228 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  Anti-CD3 epsilon antibody [SP7] - BSA and Azide free ab205228 Anti-CD3 antibody [SP7] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  Panel A: merged staining of anti-CD31 (gray; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD19 (red; Opal™570) on Formalin/PFA-fixed paraffin-embedded sections of human spleen. Secondary antibody was Opal Polymer HRP Ms + Rb, nuclear counterstain was DAPI.
  Panel B: anti-CD3 epsilon stained on T cells with ab16669 at 1/500 dilution
  Panel C: anti-CD19 stained on B cells with Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 at 1/5000 dilution
  Panel D: anti-CD31 stained on endothelial cells with Anti-CD31 antibody [EPR3094] - BSA and Azide free ab207090 at 1/500 dilution
  
  The section was incubated in three rounds of staining: in the order of Anti-CD31 antibody [EPR3094] - BSA and Azide free ab207090 and ab16669 for 30 mins, then Anti-CD19 antibody [SP291] - BSA and Azide free ab237772 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins"

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of tonsil using rabbit anti-CD3 epsilon antibody
  
  Immunohistochemical analysis of formalin fixed paraffin (FFPE) embedded tonsil labelling CD3 epsilon with ab16669 at a dilution of 1/600. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an ChromoMap DAB (RUO) IHC Detection Kit with anti rabbit HQ and anti HQ HRP. Heat mediated antigen retrieval was conducted for 24 min with DISCOVERY cell conditioning solution (CC1)  100°C, pH 8.5. ab16669 was incubated at 37°C for 16 min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of mouse spleen using rabbit anti-CD3 epsilon antibody
  
  

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining CD21+CD35 with Anti-CD21+CD35 antibody [RM2062] ab318999 at a 1:2000 (0.255 ug/ml) dilution, Anti-CD19 antibody [EPR23174-145] ab245235 anti-CD19 used at 1:1000 (0.444 ug/ml) dilution and ab16669 anti-CD3 used at a 1:150 (0.06 ug/ml) dilution.

  Panel A: merged staining of anti-CD21+CD35 (green; Opal™520), anti-CD19 (magenta; Opal™690) and anti-CD3 (yellow; Opal™570) on mouse spleen.
  
  Panel B: anti-CD21+CD35 staining B lymphocytes in mouse spleen.
  
  Panel C: anti-CD19 staining B lymphocytes in mouse spleen.
  
  Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-CD21+CD35 antibody [RM2062] ab318999, Anti-CD19 antibody [EPR23174-145] ab245235 and ab16669 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of mouse spleen using rabbit anti-CD3 epsilon antibody
  
  

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining CD272/BTLA with Anti-CD272/BTLA antibody [EPR29068-115] ab317827 at a 1:1000 (0.44 ug/ml) dilution, Anti-CD272/BTLA antibody [EPR29068-115] ab317827 anti-BTLA used at 1:100 (4.92 ug/ml) dilution and ab16669 anti-CD3 used at a 1:150 (0.06 ug/ml) dilution.

  Panel A: merged staining of anti-CD19 (magenta; Opal™690), anti-BTLA (green; Opal™520) and anti-CD3 (yellow; Opal™570) on mouse spleen.
  
  Panel B: anti-CD19 staining B lymphocytes in mouse spleen.
  
  Panel C: anti-BTLA staining B lymphocytes and T lymphocytes in mouse spleen.
  
  Panel D: anti-CD3 staining T lymphocytes in mouse spleen.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-CD19 antibody [EPR23174-145] ab245235, Anti-CD272/BTLA antibody [EPR29068-115] ab317827 and ab16669 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human tonsil using rabbit anti-CD3 epsilon antibody
  
  

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue staining C3a R with Anti-C3a R antibody [EPR28765-1] ab317321 at a 1:1000 (0.52 ug/ml) dilution, Anti-CD14 antibody [SP192] ab183322 anti-CD14 used at 1:100 (1.22 ug/ml) dilution and ab16669 anti-CD3 epsilon used at a 1:150 (0.06 ug/ml) dilution.

  Panel A: merged staining of anti-C3AR1 (green; Opal™520) anti-CD14 (red; Opal™570) anti-CD3 epsilon (magenta; Opal™690) on human tonsil.
  Panel B: anti-C3AR1 stained on immune cells.
  Panel C: anti-CD14 stained on macrophages.
  Panel D: anti-CD3 epsilon stained on T lymphocytes.
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-C3a R antibody [EPR28765-1] ab317321, Anti-CD14 antibody [SP192] ab183322 and ab16669 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

  Nuclear counter stain with DAPI.

  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human tonsil using rabbit anti-CD3 epsilon antibody
  
  Chromogenic multiplex immunohistochemical staining of FFPE normal human tonsil tissue. Anti-Ki67 antibody [SP6] ab16667 anti-Ki67 DAB chromogen. ab16669 anti-CD3 epsilon purple chromogen and Anti-CD68 antibody [SP251] ab192847 anti-CD68 teal chromogen plus haematoxylin II counterstain. Chromogenic immunostaining was performed on a Roche Ventana Benchmark Ultra instrument. The section was deparaffinised and incubated with CC1 solution for 24min 100oC. Following this with 3 rounds of staining in the order of Anti-Ki67 antibody [SP6] ab16667 (1/500 dilution), Anti-CD68 antibody [SP251] ab192847 (1/4000 dilution) and ab16669 (1/1000 dilution). Between rounds of staining antibody denaturation was conducted using Ultra CC2 solution for 8min at 100oC to avoid cross reactivity. Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with Chromomap DAB kit Discovery purple or Discovery teal chromogens and haematoxylin II counterstain.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of human thymus using rabbit anti-CD3 epsilon antibody
  
  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human thymus tissue labeling CD3 epsilon with ab16669 at 1/500 dilution, LY75/DEC-205 with Anti-LY75/DEC-205 antibody [EPR5233] - BSA and Azide free ab208649 at 1/15000, and CD68 with Anti-CD68 antibody [EPR20545] ab213363 at 1/500 dilution.
  
  Panel A: merged staining of anti-CD68 (magenta; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-LY75/DEC-205 (red; Opal™570) on human thymus.
  Panel B: anti-CD3 epsilon stained on T cells.
  Panel C: anti-LY75/DEC-205 stained on thymic cortical epithelium and dendritic cells.
  Panel D: anti-CD68 stained on macrophages.
  
  Sections  were treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins before antibody incubation. The section was incubated in three rounds of staining: in the order of Anti-CD68 antibody [EPR20545] ab213363, ab16669, and Anti-LY75/DEC-205 antibody [EPR5233] - BSA and Azide free ab208649 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  DAPI was used as a nuclear counterstain.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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  Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7] (ab16669)

  CD3 epsilon immunohistochemistry staining of rat spleen using rabbit anti-CD3 epsilon antibody
  
  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat spleen tissue labeling Sialoadhesin/CD169, CD3 epsilon and CD20 with Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and Anti-CD20 antibody [SP32] - BSA and Azide free ab236434 at 1:5000 dilution.
  
  Panel A: merged staining of anti-Sialoadhesin/CD169 (red; Opal™690), anti-CD3 epsilon (green; Opal™520) and anti-CD20 (gray; Opal™570) on rat spleen.
  Panel B: anti-Sialoadhesin/CD169 stained on macrophages.
  Panel C: anti-CD3 epsilon stained on T cells.
  Panel D: anti-CD20 stained on B cells.
  
  The section was incubated in three rounds of staining: in the order of Anti-Sialoadhesin/CD169 antibody [EPR27102-11] ab312840, ab16669, and Anti-CD20 antibody [SP32] - BSA and Azide free ab236434 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.