重组Anti-CD3 epsilon抗体[SP7] (ab16669)

Multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections of human tonsil tissue labeling CD-161 with ab302564 at 1/100 dilution (4.96 μg/mL), NKG2A with ab260035 at 1/2000 dilution ( 0.283 μg/ml) and CD3 with ab16669 at 1/2000 dilution (0.0675 µg/mL).

Panel A: merged staining of anti-CD3 (ab16669, grey; Opal™690), anti-CD161(ab302564, red; Opal™570) and anti-NKG2A (ab260035, green; Opal™520) on human NK-T lymphoma.
Panel B: anti-CD161 staining only.
Panel C: anti-NKG2A staining only.
Panel D: anti-CD3 staining only.

The section was incubated in three rounds of staining: in the order of ab16669 for 30 mins, ab302564 for 30 mins and ab260035 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Nuclear stained with DAPI.

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

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.

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.

Chromogenic multiplex immunohistochemical staining of FFPE normal human tonsil tissue. Ab16667, anti-Ki67 DAB chromogen. Ab16669, anti-CD3 epsilon purple chromogen and 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, 100°C. Following this, with 3 rounds of staining in the order of ab16667 (1/500), ab192847 (1/4000) ab16669 (1/1000). Between rounds of staining, antibody denaturation was conducted using Ultra CC2 solution for 8min at 100°C 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.

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 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 ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilution

The section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then 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.

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 ab237772 at 1/5000 dilution
Panel D: anti-CD68 stained on macrophages with ab213363 1/500 dilution

The section was incubated in three rounds of staining: in the order of ab213363 and ab16669 for 30 mins, then 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.

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 ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells and immune cell subsets with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then 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

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 ab237772 at 1/5000 dilution
Panel D: anti-CD31 stained on endothelial cells with ab207090 at 1/500 dilution
The section was incubated in three rounds of staining: in the order of ab207090 and ab16669 for 30 mins, then 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

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 ab208649 at 1/15000, and CD68 with 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 ab213363, ab16669, and 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.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Rat spleen tissue labeling Sialoadhesin/CD169, CD3 epsilon and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and 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 ab312840, ab16669, and 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.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Mouse spleen tissue labeling Sialoadhesin/CD169, CD3 epsilon and CD20 with ab312840 at 1/100 dilution, ab16669 at 1:150 dilution and 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 ab312840, ab16669, and 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.

Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 epsilon (ab16669, green; Opal™520) and anti-CD68 (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 ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and 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.

Fluorescence multiplex immunohistochemical analysis of the human duodenum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-liver FABP (ab240401, gray; Opal™690), anti-CD3 epsilon (ab16669, green; Opal™520) and anti-CD68 (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 ab240401 (1/8000 dilution), ab16669 (1/150 dilution), and 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.

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, 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 ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 epsilon (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (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 ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and 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.

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) (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.

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.

See Abreview

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

IHC using multi-spectral imaging on human lymph node (A-C) obtained from men with mCRPC. A) H+E staining and B) single-color images (plus nuclear stain; DAPI) of CD3 epsilon (ab16669), CD8 (ab101500), CD163, PD-L1, cytokeratin (CK), DAPI and C) merged.  H+E stating at 20X magnification; multi-spectral images 200X magnification.

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.

See Abreview

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) in Jurkats cells (green) compare to negative control of rabbit IgG (blue).

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.

See Abreview

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.