CD3 Recombinant Rabbit Monoclonal Antibody [JE80-02]

Western blot analysis of CD3 on Jurkat cell lysates with Rabbit anti-CD3 antibody (HA720082) at 0.5μg/ml dilution.

Lysates/proteins at 10 µg/Lane.

Predicted band size: 23 kDa
Observed band size: 23 kDa

Exposure time: 2 minutes;

15% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA720082) at 0.5μg/ml dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Fluorescence multiplex immunohistochemical analysis of Tertiary Lymphoid Structures in Human Small Cell Lung Cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-PD-L1 (HA721176, cyan), anti-CD56 (ET1702-43, magenta) and anti-CD3 (HA720082, yellow) on tertiary lymphoid structures. Panel B: anti- CD20 stained on B cells. Panel C: anti-PD-L1 stained on dendritic cells and macrophages cells. Panel D: anti-CD56 stained on NKT cells. Panel E: anti-CD3 stained on T cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in four rounds of staining: in the order of HA721138 (1/1,500 dilution), HA721176 (1/1,000 dilution), ET1702-43 (1/1,000 dilution), and HA720082 (1/500 dilution) for 20 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) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of the human non-small cell lung cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-CD68 (HA601115, gray), anti-PD-L1 (HA721176, cyan), anti-panCK (HA601138, magenta) and anti-CD3 (HA720082, yellow) on human non-small cell lung cancer. Panel B: anti- CD20 stained on B cells. Panel C: anti-CD68 stained on macrophage M1 and macrophage M2. Panel D: anti-PD-L1 stained on dendritic cells and macrophages cells. Panel E: anti-panCK stained on cancer cells. Panel F: anti-CD3 stained on T cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in five rounds of staining: in the order of HA721138 (1/1,500 dilution), HA601115 (1/2,000 dilution), HA721176 (1/1,000 dilution), HA601138 (1/3,000 dilution), and HA720082 (1/500 dilution) for 20 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) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of the human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, red), anti-αSMA (ET1607-53, gray), anti-CD11b (ET1706-04, cyan), anti-panCK (HA601138, magenta) and anti-CD3 (HA720082, yellow) on human gastric cancer. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-αSMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-CD11b stained on myeloid cells. Panel E: anti-panCK stained on cancer cells. Panel F: anti-CD3 stained on T cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in five rounds of staining: in the order of M1511-8 (1/1,000 dilution), ET1607-53 (1/2,000 dilution), ET1706-04 (1/1,000 dilution), HA601138 (1/3,000 dilution), and HA720082 (1/500 dilution) for 20 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) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of the human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-Ki67 (HA721115, red), anti-CD31 (M1511-8, green), anti-CD3 (HA720082, cyan), anti-panCK (HA601138, magenta) and anti-αSMA (ET1607-53, yellow) on human gastric cancer. Panel B: anti- Ki67 stained on cells in G1, S, G2 and M phases of cell cycle. Panel C: anti-CD31 stained on the endothelial cells. Panel D: anti-CD3 stained on T cells. Panel E: anti-panCK stained on cancer cells. Panel F: anti-αSMA stained on cancer-associated fibroblasts and smooth muscle cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in five rounds of staining: in the order of HA721115 (1/2,000 dilution), M1511-8 (1/1,000 dilution), HA720082 (1/500 dilution), HA601138 (1/3,000 dilution), and ET1607-53 (1/2,000 dilution) for 20 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) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD11b (ET1706-04, Red), anti-CD3 (HA720082, Green) and anti-CD31 (M1511-8, Yellow) on human gastric cancer. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of ET1706-04 (1/1,000 dilution), HA720082 (1/500 dilution) and M1511-8 (1/1,000 dilution) for 20 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) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.

Immunohistochemical analysis of paraffin-embedded human lymph nodes tissue with Rabbit anti-CD3 antibody (HA720082) at 1/600 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA720082) at 1/600 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-CD3 antibody (HA720082) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA720082) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunocytochemistry analysis of Jurkat cells labeling CD3 with Rabbit anti-CD3 antibody (HA720082) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CD3 antibody (HA720082) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunofluorescence analysis of paraffin-embedded human lymph nodes tissue labeling CD3 with Rabbit anti-CD3 antibody (HA720082) at 1/100 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA720082, green) at 1/100 dilution overnight at 4 ℃, washed with PBS.

Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

CD3 was immunoprecipitated from 1mg/ml Jurkat whole cell lysate with HA720082 at 2ug/ml dilution. Western blot was performed from the immunoprecipitate using HA720082 at 1ug/ml dilution. Goat anti-Rabbit IgG-HRP antibody (HA1001) was used as the secondary antibody at 1/300,000 dilution.

Lane 1: Jurkat whole cell lysate 5 μg (Input).
Lane 2: HA720082 IP in Jurkat whole cell lysate.
Lane 3: Rabbit monoclonal IgG instead of HA720082 in Jurkat whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.