CD4 Recombinant Rabbit Monoclonal Antibody [ST0488]
cat.: ET1609-52
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human
Applications: WB, IF-Cell, IF-Tissue, IHC-P, FC, mIHC
Clonality: Monoclonal
Clone number: ST0488
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 51 kDa
Isotype: IgG
Immunogen: Recombinant protein within Human CD4 aa 196-416 / 458.
Positive control: U937 cell lysate, THP-1 cell lysate, THP-1, human tonsil tissue, human spleen tissue, human lymph nodes tissue, human liver tissue, human prostate cancer, human cervical cancer.
Subcellular location: Cell membrane.
Recommended Dilutions:
  WB
  IF-Cell
  IF-Tissue
  IHC-P
  FC
  mIHC
1:1,000-1:2,000
1:50-1:200
1:50-1:200
1:400-1:800
1:500-1:1,000
1:800-1:1,000
Uniprot #: SwissProt: P01730 Human
Alternative names: CD 4 CD4 (L3T4) CD4 CD4 antigen (p55) CD4 antigen CD4 molecule CD4 receptor CD4+ Lymphocyte deficiency, included CD4_HUMAN CD4mut L3T4 Leu3 Ly-4 Lymphocyte antigen CD4 MGC165891 OTTHUMP00000238897 p55 T cell antigen T4 T cell antigen T4/LEU3 T cell differentiation antigen L3T4 T cell OKT4 deficiency, included T cell surface antigen T4/Leu 3 T cell surface antigen T4/Leu3 T cell surface glycoprotein CD4 T-cell surface antigen T4/Leu-3 T-cell surface glycoprotein CD4 W3/25 W3/25 antigen
Images
ET1609-52_1.jpg Fig1: Fluorescence multiplex immunohistochemical analysis of tertiary lymphoid structures in human prostate cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-CD21 (HA721163, cyan) and anti-CD4 (ET1609-52, yellow) on tertiary lymphoid structures. Panel B: anti- CD20 stained on B cells. Panel C: anti-CD21 stained on naive B-cell, memory B-cell and plasma cells. Panel D: anti-CD4 stained on helper T cells and Treg 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 three rounds of staining: in the order of HA721138 (1/1,500 dilution), HA721163 (1/1,000 dilution), and ET1609-52 (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 Olympus VS200 Slide Scanner.
ET1609-52_2.jpg Fig2: Fluorescence multiplex immunohistochemical analysis of tertiary lymphoid structures in human cervical cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-Granzyme B (HA500252, magenta), anti-CD4 (ET1609-52, yellow) on tertiary lymphoid structures. Panel B: anti- Granzyme B stained on cytotoxic NK cells and dendritic cells. Panel C: anti-CD4 stained on helper T cells and Treg 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 three rounds of staining: in the order of HA500252 (1/200 dilution), ET1609-52 (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 Olympus VS200 Slide Scanner.
ET1609-52_3.jpg Fig3: Fluorescence multiplex immunohistochemical analysis of Human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD14 (ET1610-85, Red), anti-CD4 (ET1609-52, Green), anti-CD57 (HA601114, White), anti-CD15 (HA721246, Cyan)and anti-Tryptase (ET1610-64, Magenta) on tonsil. Panel B: anti- CD14 stained on monocytes. Panel C: anti-CD4 stained on helper T cells and Treg cells. Panel D: anti-CD57 stained on NK cells and T cells. Panel E: CD15 stained on granulocytes and monocytes. Panel F: anti-Tryptase stained on Mast 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 ET1610-85 (1/800 dilution), ET1609-52 (1/800 dilution), HA601114 (1/1,000 dilution), HA721246 (1/500 dilution), and ET1610-64 (1/3,000 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.
ET1609-52_4.jpg Fig4: Fluorescence multiplex immunohistochemical analysis of human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD68 (HA601115, Red), anti-BCL6 (HA601083, Yellow) and anti-CD4 (ET1609-52, Green) on tonsil. 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 HA601115 (1/2,000 dilution), HA601083 (1/200 dilution) and ET1609-52 (1/800 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.
ET1609-52_5.jpg Fig5: Western blot analysis of CD4 on different lysates with Rabbit anti-CD4 antibody (ET1609-52) at 1/1,000 dilution.

Lane 1: THP-1 cell lysate
Lane 2: U937 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 51 kDa
Observed band size: 55 kDa

Exposure time: 1 minute 30 seconds;

10% 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 (ET1609-52) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
ET1609-52_6.jpg Fig6: Western blot analysis of CD4 on different lysates with Rabbit anti-CD4 antibody (ET1609-52) at 1/2,000 dilution.

Lane 1: THP-1 WT cell lysate
Lane 2: THP-1 CD4 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 51 kDa
Observed band size: 55 kDa

Exposure time: 30 seconds;
ECL: Ori Supersensitive

4-20% SDS-PAGE gel.

ET1609-52 was shown to specifically react with CD4 in THP-1 WT cells. Weakened band was observed when THP-1 CD4 KD sample was tested. THP-1 WT and THP-1 CD4 KD samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1609-52, 1/2,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
ET1609-52_7.jpg Fig7: Immunocytochemistry analysis of THP-1 cells labeling CD4 with Rabbit anti-CD4 antibody (ET1609-52) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-CD4 antibody (ET1609-52) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. 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.
ET1609-52_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-CD4 antibody (ET1609-52) at 1/800 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 (ET1609-52) at 1/800 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.
ET1609-52_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-CD4 antibody (ET1609-52) at 1/400 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 (ET1609-52) at 1/400 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.
ET1609-52_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded human lymph nodes tissue with Rabbit anti-CD4 antibody (ET1609-52) at 1/400 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 (ET1609-52) at 1/400 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.
ET1609-52_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-CD4 antibody (ET1609-52) at 1/800 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 (ET1609-52) at 1/800 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.
ET1609-52_12.jpg Fig12: Flow cytometric analysis of THP-1 cells labeling CD4.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (ET1609-52, 1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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