Anti- IL-4 antibody [EPR29904-542]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti- IL-4 antibody [EPR29904-542] (AB325181)

Flow cytometric analysis of Mouse CD4+ PBMC cultured under Th2-polarizing conditions for 4 days then treated with 10ng/mL PMA, 500ng/mL Ionomycin and 300ng/ml BFA for 4 hours (Lower left and right) / Mouse CD4+ PBMC cultured under Th2-polarizing conditions for 4 days (Upper left and right) cells labelling IL-4 with ab325181 at 1/5000 dilution (0.01ug) / Upper right and Lower 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® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells were co-stained with anti-GATA-3 antibody conjugated to Alexa Fluor®488.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti- IL-4 antibody [EPR29904-542] (AB325181)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse PBMC (mouse peripheral blood mononuclear cell) cells labelling IL-4 with ab325181 at 1/50 (10.04 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased cytoplasmic staining in a subset of Th2 differentiated mouse PBMC treated with Phorbol-12-myristate-13-acetate (10 ng/mL) and Ionomycin (500 ng/mL) and Brefeldin A (300 ng/mL) for 4 hr (shown in green). 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 CD4 mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (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

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Immunocytochemistry/ Immunofluorescence - Anti- IL-4 antibody [EPR29904-542] (AB325181)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) cells labelling IL-4 with ab325181 at 1/200 (2.51 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased cytoplasmic staining in a subset of EL4 cells (shown in green) treated with PHA (10 ug/mL) and Phorbol-12-myristate-13-acetate (10 ng/mL) for 48 hr with addition of brefeldin A (500 ng/mL) for the last 16 hr. 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).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 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.

• WB

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Western blot - Anti- IL-4 antibody [EPR29904-542] (AB325181)

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

The identity of the bands above 75 kDa are unknown.

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

All lanes:

Western blot - Anti- IL-4 antibody [EPR29904-542] (ab325181) at 1/1000 dilution

Lane 1:

Untreated EL4.IL-2 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

Lane 2:

EL4.IL-2 treated with 10µg/ml PHA and 10ng/ml PMA for 48 hours, 500 ng/ml BFA was then added for overnight whole cell lysate at 20 µg

Secondary

All lanes:

Western blot at 1/20000 dilution

Observed band size: 15 kDa,36 kDa

false

Exposure time: 70s