Recombinant Anti-CD4 antibody [RM1013] (ab288724)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling CD4 with ab288724 at 1/1000 (0.55 μg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human tonsil. The section was incubated with ab288724 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling CD4 with ab288724 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in THP-1 cell line. Negative control: K-562. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

All lanes : Anti-CD4 antibody [RM1013] (ab288724) at 1/1000 dilution

Lane 1 : THP-1 (Human monocytic leukemia monocyte) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 3 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 4 : Human lymphoma lysate
Lane 5 : Mouse thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

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


Exposure time: 70 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Negative control: K-562.

CD4 was immunoprecipitated from 0.35 mg THP-1 (Human monocytic leukemia monocyte) whole cell lysate with ab288724 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab288724 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (Human monocytic leukemia monocyte) whole cell lysate 10 μg

Lane 2: ab288724 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab288724 in THP-1 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse thymus (fresh) tissue labeling CD4 with ab288724 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (Green). Positive staining on mouse thymus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

IHC image of CD4 staining in a section of frozen human tonsil* performed on a Leica Biosystems BOND^® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab288724, 0.5 ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling CD4 with ab288724 at 1/1000 (0.55 μg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human spleen. The section was incubated with ab288724 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling CD4 with ab288724 at 1/1000 (0.55 μg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on the mouse spleen. The section was incubated with ab288724 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized Mouse thymus cell cells labelling CD4 with ab288724 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in mouse thymus cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

CD4 was immunoprecipitated from 0.35 mg Mouse thymus lysate with ab288724 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab288724 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse thymus lysate 10μg

Lane 2: ab288724 IP in Mouse thymus lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab288724 in Mouse thymus lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 23 seconds