Anti-CD5 antibody [RM1352]

• Flow Cyt

Lab

Flow Cytometry - Anti-CD5 antibody [RM1352] (AB325643)

Flow cytometric analysis of MOLT-4 (human lymphoblastic leukemia T lymphoblast) (Right) HeLa (human cervical adenocarcinoma epithelial cell) (Left) cells labelling CD5 with ab325643 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Negative control : HeLa (PMID : 15187131).

• Flow Cyt

Lab

Flow Cytometry - Anti-CD5 antibody [RM1352] (AB325643)

Flow cytometric analysis of Human PBMC (human peripheral blood mononuclear cell) cells labelling CD5 with ab325643 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti human CD3 conjugated to Alexa Fluor®647.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD5 antibody [RM1352] (AB325643)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling CD5 with ab325643 at 1/250 (1.92 µg/ml) 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 human PBMCs(shown in green), which is co-stained with CD3 marker. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-human CD3 mouse monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 dilution (Magenta). 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD5 antibody [RM1352] (AB325643)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MOLT-4 (human lymphoblastic leukemia T lymphoblast) HeLa (human cervical adenocarcinoma epithelial cell) cells labelling CD5 with ab325643 at 1/250 (1.92 µg/ml) 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 MOLT-4 cells and no staining in HeLa cells(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Negative control : HeLa (PMID : 15187131). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325643 at 1/250 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human cervical cancer.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on immune cells of human colon.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-CD5 antibody [RM1352] (AB325643)

CD5 was immunoprecipitated from 0.35 mg MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate with ab325643 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325643 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate
Lane 2 : ab325643 IP in MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325643 in MOLT-4 whole cell lysate

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

Exposure time : 76 seconds.

All lanes:

Immunoprecipitation - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 10 µg

Lane 2:

ab325643 at 1/30 IP in MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325643 in MOLT-4 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 67 kDa

false

Exposure time: 76s

• Flow Cyt

Lab

Flow Cytometry - Anti-CD5 antibody [RM1352] (AB325643)

Flow cytometric analysis of EL4 (mouse lymphoma T lymphocyte) (Right) A20 (mouse reticulum sarcoma B lymphocyte) (Left) cells labelling CD5 with ab325643 at 1/500 dilution (0.1ug) / Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Negative control : A20.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Mouse lung cancer tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung cancer.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt

Lab

Flow Cytometry - Anti-CD5 antibody [RM1352] (AB325643)

Flow cytometric analysis of Mouse PBMC cells labelling CD5 with ab325643 at 1/500 dilution (0.1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti mouse CD3 conjugated to Alexa Fluor®647.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD5 antibody [RM1352] (AB325643)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) A20 (mouse reticulum sarcoma B lymphocyte) cells labelling CD5 with ab325643 at 1/250 (1.92 µg/ml) 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 EL4 cells and no staining in A20 cells(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Negative control : A20. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325643 at 1/250 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD5 antibody [RM1352] (AB325643)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse PBMC (mouse peripheral blood mononuclear cell) cells labelling CD5 with ab325643 at 1/250 (1.92 µg/ml) 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 PBMCs(shown in green), which is co-stained with CD3 marker. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-mouse CD3 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 dilution (Magenta). 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-CD5 antibody [RM1352] (AB325643)

CD5 was immunoprecipitated from 0.35 mg EL4 (mouse lymphoma T lymphocyte) whole cell lysate with ab325643 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325643 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 2 : ab325643 IP in EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325643 in EL4 whole cell lysate

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

Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 10 µg

Lane 2:

ab325643 at 1/30 IP in EL4 (mouse lymphoma T lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325643 in EL4 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 67 kDa

false

Exposure time: 32s

• WB

Lab

Western blot - Anti-CD5 antibody [RM1352] (AB325643)

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

Negative control : HeLa

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 2472278; PMID : 23626783).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 2:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 3:

CCRF-CEM (human peripheral blood T lymphoblast) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell 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 1/20000 dilution

Observed band size: 67 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD5 antibody [RM1352] (AB325643)

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

Negative control : Skeletal muscle

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Observed band size: 67 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD5 antibody [RM1352] (AB325643)

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

Negative control : Skeletal muscle

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

Mouse thymus tissue lysate at 20 µg

Lane 2:

Mouse lymph node tissue lysate at 20 µg

Lane 3:

Mouse skeletal muscle tissue 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 1/20000 dilution

Observed band size: 67 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD5 antibody [RM1352] (AB325643)

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

Negative control : A20 (PMID : 19520434).

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-CD5 antibody [RM1352] (ab325643) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 2:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell 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 1/20000 dilution

Observed band size: 67 kDa,36 kDa

true

Exposure time: 103s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human skeletal muscle.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD5 antibody [RM1352] (AB325643)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling CD5 with ab325643 at 1/1000 (0.48 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse skeletal muscle.

The primary antibody was incubated 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins