Anti-CD3 epsilon antibody [SP7], prediluted

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human colon tissue staining CD3 with ab21703 at a 1/150 (0.06 ug/ml) dilution, ab318205 anti-TCR beta used at 1/500 (1.036 ug/ml) dilution and ab64088 anti-CD20 used at a 1/50 (0.2 ug/ml) dilution.

Panel A : merged staining of anti-CD3 (magenta; Opal™690), anti-TCR beta (green; Opal™520) and anti-CD20 (yellow; Opal™570) on human colon.
Panel B : anti-CD3 staining T lymphocytes in human colon.
Panel C : anti-TCR beta staining T lymphocytes in human colon.
Panel D : anti-CD20 staining B lymphocytes in human colon.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab21703, ab318205 and ab64088 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human spleen tissue staining CD3 with ab21703 at a 1/150 (0.06 ug/ml) dilution, ab318205 anti-TCR beta used at 1/500 (1.036 ug/ml) dilution and ab64088 anti-CD20 used at a 1/50 (0.2 ug/ml) dilution.

Panel A : merged staining of anti-CD3 (magenta; Opal™690), anti-TCR beta (green; Opal™520) and anti-CD20 (yellow; Opal™570) on human spleen.
Panel B : anti-CD3 staining T lymphocytes in human spleen.
Panel C : anti-TCR beta staining T lymphocytes in human spleen.
Panel D : anti-CD20 staining B lymphocytes in human spleen.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab21703, ab318205 and ab64088 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

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 data was developed using the undiluted version of this antibody (ab16669).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Immunohistochemical analysis of paraffin-embedded Human tonsil labeling CD3 epsilon with ab21703 at 1/150 (7 μg/ml). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab21703 for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. DAB was used as the chromogen. Counterstained with Hematoxylin and mounted with DPX.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue staining CD3 with ab21703 at a 1/150 (0.06 ug/ml) dilution, ab318205 anti-TCR beta used at 1/500 (1.036 ug/ml) dilution and ab64088 anti-CD20 used at a 1/50 (0.2 ug/ml) dilution.

Panel A : merged staining of anti-CD3 (magenta; Opal™690), anti-TCR beta (green; Opal™520) and anti-CD20 (yellow; Opal™570) on human tonsil.
Panel B : anti-CD3 staining T lymphocytes in human tonsil.
Panel C : anti-TCR beta staining T lymphocytes in human tonsil.
Panel D : anti-CD20 staining B 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 ab21703, ab318205 and ab64088 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat spleen tissue staining CD3 with ab21703 at a 1/150 (0.06 ug/ml) dilution, ab318205 anti-TCR beta used at 1/500 (1.036 ug/ml) dilution and ab64088 anti-CD20 used at a 1/50 (0.2 ug/ml) dilution.

Panel A : merged staining of anti-CD3 (magenta; Opal™690), anti-TCR beta (green; Opal™520) and anti-CD20 (yellow; Opal™570) on rat spleen.
Panel B : anti-CD3 staining T lymphocytes in rat spleen.
Panel C : anti-TCR beta staining T lymphocytes in rat spleen.
Panel D : anti-CD20 staining B lymphocytes in rat spleen.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab21703, ab318205 and ab64088 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.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse spleen tissue staining CD3 with ab21703 at a 1/150 (0.06 ug/ml) dilution, ab318205 anti-TCR beta used at 1/500 (1.036 ug/ml) dilution and ab64088 anti-CD20 used at a 1/50 (0.2 ug/ml) dilution.

Panel A : merged staining of anti-CD3 (magenta; Opal™690), anti-TCR beta (green; Opal™520) and anti-CD20 (yellow; Opal™570) on mouse spleen.
Panel B : anti-CD3 staining T lymphocytes in mouse spleen.
Panel C : anti-TCR beta staining T lymphocytes in mouse spleen.
Panel D : anti-CD20 staining B 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 ab21703, ab318205 and ab64088 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.

• WB

Lab

Western blot - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

This data was developed using ab16669, the same antibody clone in a different buffer formulation.

Negative control : U-937, Raji, brain.

All lanes:

Western blot - Anti-CD3 epsilon antibody [SP7] (<a href='/en-us/products/primary-antibodies/cd3-epsilon-antibody-sp7-ab16669'>ab16669</a>) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

MOLT-4 (Human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 3:

U-937 (Human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 4:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 5:

EL4.IL-2 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 6:

Mouse thymus lysate at 20 µg

Lane 7:

Mouse brain lysate at 20 µg

Lane 8:

Rat thymus lysate at 20 µg

Lane 9:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 23 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD3 epsilon antibody [SP7], prediluted (AB21703)

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 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.

This data was developed using the undiluted version of this antibody (ab16669).

All lanes:

Western blot - Anti-CD3 epsilon antibody [SP7], prediluted (ab21703) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 19 kDa

Observed band size: 23 kDa

false