Anti-IRF4 antibody [EPR28687-87]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining in human pancreas.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in Human Hodgkin's lymphoma.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded A Ramos (human Burkitt's lymphoma B lymphocyte) cell pellets; B Jurkat (human T cell leukemia T lymphocyte from peripheral blood) cell pellets. tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in A : Ramos cell pellets, nearly no staining in B : Jurkat cell pellets.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human tonsil.
The section was incubated with ab315394 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 (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC (human peripheral blood mononuclear cell) treated with 10ug/ml PHA for 3days (Red) / Untreated human PBMC (Dotted-red) labelling IRF4 with ab315394 at 1/500 dilution (0.1 ug)/Red and dotted-red, compared with a Rabbit monoclonal IgG (ab172730)  (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

Cells are gated on CD4 positive cells. Cells were stained with anti-CD4 conjugated to Alexa Fluor®647. Fixed with 2% PFA for 10 min followed by intracellularly staining with primary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized J558 (mouse B lymphocyte lymphoblast) cells labelling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing mainly nuclear staining in J558 cells and showing weak nuclear staining in EL4 cells.
Low expression : EL4 (PMID : 24711583).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) 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 (2 ug/ml) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining in rat pancreas.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining in mouse pancreas.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse spleen.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in rat spleen.
The section was incubated with ab315394 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Immunohistochemical analysis of paraffin-embedded Mouse Burkitt's lymphoma tissue labeling IRF4 with ab315394 at 1/100 (5.12 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse Burkitt's lymphoma.
The section was incubated with ab315394 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 (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Mouse PBMC (mouse peripheral blood mononuclear cell) treated with 10ug/ml PHA for 3days (Red) / Untreated Mouse PBMC (Dotted-red), labelling IRF4 with ab315394 at 1/500 dilution (0.1 ug)/Red and dotted-red, compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

Cells are gated on CD4 positive cells. Cells were stained with anti-CD4 conjugated to Alexa Fluor®647. Then fixed with 2% PFA for 10 min followed by intracellularly staining with primary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IRF4 antibody [EPR28687-87] (AB315394)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized EL4 (mouse lymphoma T lymphocyte, Left) / J558 (mouse B lymphocyte lymphoblast, Right) cells labelling IRF4 with ab315394 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

Low expression : EL4.

• IP

Supplier Data

Immunoprecipitation - Anti-IRF4 antibody [EPR28687-87] (AB315394)

IRF4 was immunoprecipitated from 0.35 mg Mouse lymph node tissue lysate with ab315394 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315394 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-IRF4 antibody [EPR28687-87] (ab315394) at 1/1000 dilution

Lane 1:

Mouse lymph node tissue lysate

Lane 2:

ab315394 at 1/30 IP in Mouse lymph node tissue lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab315394 in Mouse lymph node tissue 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

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-IRF4 antibody [EPR28687-87] (AB315394)

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

Low expression : Jurkat(PMID : 10714679), EL4(PMID : 24711583)

Lower bands could be ubiquitin mediated protein degradation. (PMID : 23658517)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IRF4 antibody [EPR28687-87] (ab315394) at 1/1000 dilution

Lane 1:

Ramos (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

J558 (mouse B lymphocyte lymphoblast) whole cell lysate at 20 µg

Lane 4:

EL4 (mouse lymphoma T 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 1/100000 dilution

Observed band size: 35 kDa,51 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-IRF4 antibody [EPR28687-87] (AB315394)

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

Lower bands could be ubiquitin mediated protein degradation. (PMID : 23658517).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IRF4 antibody [EPR28687-87] (ab315394) at 1/1000 dilution

Lane 1:

Mouse lymph node tissue lysate at 20 µg

Lane 2:

Rat spleen tissue lysate at 20 µg

Lane 3:

Rat lymph node 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 1/100000 dilution

Observed band size: 51 kDa,35 kDa,36 kDa

false