Human peripheral blood lymphocytes stained with unpurified ab133616 (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 (unpurified ab133616, 1/100 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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 same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with purified ab133616 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

This data was developed using the same antibody clone in a different buffer formulation (ab133616).
Immunocytochemistry analysis of THP-1 (Human monocytic leukemia monocyte) labeling CD4 with purified ab133616 at 1/100 dilution. Cells were fixed with 100% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.30 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

Imaging Mass Cytometry™ (IMC™) image of human tonsil tissue stained with Anti-CD4 antibody [EPR6855]. ab181724 (carrier-free antibody, purified) was metal-conjugated using a Maxpar® Antibody Labeling Kit from Fluidigm. Immunostaining was performed according to Fluidigm’s protocols. Briefly, slides were subject to deparaffinization and heat-induced epitope retrieval, followed by overnight incubation at 4°C with an antibody cocktail containing metal-tagged antibodies in blocking buffer. Slides were subsequently washed with 0.2% Triton-X and 1x PBS, counterstained with Cell-ID™ Intercalator-Ir diluted at 1/400 in 1x PBS for 30 min at room temperature, rinsed for 5 min with distilled H2O, and air-dried prior to IMC™ acquisition. IMC™ acquisition was performed using the Fluidigm Hyperion™ Imaging System.

Imaging Mass Cytometry™, IMC™, Cell-ID™, Hyperion™ and Maxpar® are trademarks of Fluidigm Canada Inc.​​

Anti-CD4 antibody [EPR6855] - BSA and Azide free (ab181724) + THP-1 (human monocytic leukemia cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 51 kDa


Exposure time: 3 minutes

This WB data was generated using the same anti-CD4 antibody clone [EPR6855] in a different buffer formulation (cat# ab133616).

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concetration: 5% NFDM/TBST

Anti-CD4 antibody [EPR6855] (ab133616)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with ab133616 at a dilution of 1:500. Heat mediated antigen retrieval was performed using AR9 antigen retrieval solution, and microwave treatment for 15 min at 20% power. Anti-Rabbit/Mouse HRP polymer (PerkinElmer Opal Polymer HRP Ms Plus Rb) was used as secondary antibody. Opal tyramide amplification was performed using Opal 520 fluorophore. Counterstained with DAPI stain. Image scanned with Vectra 3.0 and analyzed via Phenochart software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.

Clone EPR6855 (ab181724) has been successfully conjugated by Abcam. This image was generated using Anti-CD4 antibody [EPR6855] (Alexa Fluor® 488). Please refer to ab196372 for protocol details.

ab196372 staining CD4 in Jurkat cells. The cells were fixed with 100% methanol (5 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab196372 at a working dilution of 1 in 50 (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EPR6855 (ab181724) has been successfully conjugated by Abcam. This image was generated using Anti-CD4 antibody [EPR6855] (Alexa Fluor® 647). Please refer to ab196147 for protocol details.

ab196147 staining CD4 in Jurkat cells. The cells were fixed with 100%methanol and then incubated with 1%BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated overnight at +4°C with ab196147 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in Jurkat cells fixed with 4% formaldehyde.

Negative control: no staining on human cerebrum.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum showing no staining CD4 with purified ab133616 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Negative control: no staining on human pancreas.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreas showing no staining CD4 with purified ab133616 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Paraffin-embedded human spleen tissue stained for CD4 using ab133616 at 1/500 dilution in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thymoma tissue labelling CD4 with purified ab133616 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling CD4 with ab133616 at a dilution of 1/100.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling CD4 with unpurified ab133616.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD4 with unpurified ab133616.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thymoma tissue labelling CD4 with unpurified ab133616.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133616).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This IHC data was generated using the same anti-CD4 antibody clone [EPR6855] in a different buffer formulation (cat# ab133616).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with unpurified ab133616 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.