Anti-CD4 antibody [RM1013]

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD4 antibody [RM1013] (AB288724)

The BOND™ Polymer Refine Detection System on the BOND RX Automated Stainer (DS9800, Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical labelling of formalin-fixed, paraffin-embedded (FFPE) tissues. The protocol includes a peroxide block, post-primary reagent, polymer detection, DAB chromogen, and haematoxylin counterstain, all automated to minimize variability. Antigen retrieval was performed by heat-induced epitope retrieval (HIER) for 20 minutes in Tris-EDTA buffer (BOND Epitope Retrieval Solution 2, AR9640, Leica Biosystems, Wetzlar, Germany). For mouse and rabbit antibodies, the sequence comprised peroxide block (10 min), primary antibody incubation (30 min), post-primary (10 min), polymer (10 min), DAB (10 min), and haematoxylin (8 min). For rat antibodies, an anti-rat step (1 : 300 dilution, Vector Laboratories, California, USA; 20 min) was included prior to polymer application (15 min).

For mouse tissues, antigen retrieval was performed using high pH buffers (Dako, Agilent), followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide. Slides were then incubated with the primary antibody. Antigen/antibody complexes detection was carried out using a horseradish peroxidase-conjugated visualization system (Novolink Polymer, Leica). Staining was developed with 3,3′-diaminobenzidine (EnVision FLEX DAB Enhancer, Dako, Agilent), and nuclei were counterstained with hematoxylin (FLEX Hematoxylin, SM806, Dako, Agilent). Images were acquired using an Axiocam CCD camera (Zeiss) and processed with ZEN 2.1 software and Photoshop 9.0

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD4 antibody [RM1013] (AB288724)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD4 antibody [RM1013] (AB288724)

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD4 antibody [RM1013] (AB288724)

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.

• IP

Lab

Immunoprecipitation - Anti-CD4 antibody [RM1013] (AB288724)

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.

All lanes:

Immunoprecipitation - Anti-CD4 antibody [RM1013] (ab288724)

Predicted band size: 51 kDa

Observed band size: 51 kDa

false

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-CD4 antibody [RM1013] (AB288724)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD4 antibody [RM1013] (AB288724)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD4 antibody [RM1013] (AB288724)

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

• IP

Lab

Immunoprecipitation - Anti-CD4 antibody [RM1013] (AB288724)

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

All lanes:

Immunoprecipitation - Anti-CD4 antibody [RM1013] (ab288724)

Predicted band size: 51 kDa

Observed band size: 51 kDa

false

• WB

Lab

Western blot - Anti-CD4 antibody [RM1013] (AB288724)

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

Negative control : K-562.

All lanes:

Western blot - Anti-CD4 antibody [RM1013] (ab288724) at 1/1000 dilution

Lane 1:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

Human lymphoma lysate at 20 µg

Lane 5:

Mouse thymus 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 1/100000 dilution

Predicted band size: 51 kDa

Observed band size: 51 kDa

false

Exposure time: 70s