Anti-Interferon gamma antibody [EPR28352-7]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Interferon gamma antibody [EPR28352-7] (AB324874)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse spleenocytes cells labelling Interferon gamma with ab324874 at 1/250 (1.988 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in subsets of mouse splenocytes treated with Phorbol-12-myristate-13-acetate (50ng/ml) and Ionomycin (1 ug/mL) for 1hours add Brefeldin A (300ng/ml) for 3hours (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-mouse CD3 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 10ug/ml 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 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Interferon gamma antibody [EPR28352-7] (AB324874)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized mouse spleen cell treated with 50 ng/mL PMA for 1+3 hours and 1 ug/mL Ionomycin for 1+3 hours then add 300ng/ml BFA for 3 hours (Lower left and right) / Untreated control (Upper left and right) cells labelling Interferon gamma with ab324874 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.

Cells were surface stained with anti-CD3 conjugated to Alexa Fluor®488. Then fixed with 2% PFA for 10 min followed by intracellularly staining with rabbit IgG or our antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Interferon gamma antibody [EPR28352-7] (AB324874)

Flow cytometric analysis of Mouse PBMC Th1 differentiation for 4 days (Left) / Mouse PBMC Th1 differentiation for 4 days then treated with 10ng/mL PMA for 4 hours and 500ng/mL Ionomycin for 4 hours and 300ng/ml BFA for 4 hours (Right) cells labelling Interferon gamma with ab324874 at 1/5000 dilution (0.01ug) / Left and Right compared with a 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.

Cells were surface-stained with Zombie Aqua to delete dead cells, then co-stained intracellularly, using True-Nuclear™ Transcription Factor Buffer Set, with anti-T-bet conjugated to PE and our antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Interferon gamma antibody [EPR28352-7] (AB324874)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized mouse spleen cell treated with 50 ng/mL PMA for 1+3 hours and 1 ug/mL Ionomycin for 1+3 hours then add 300ng/ml BFA for 3 hours (Lower left and right) / Untreated control (Upper left and right) cells labelling Interferon gamma with ab324874 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.

Cells were surface stained with anti-CD19 conjugated to PE/CY7. Then fixed with 2% PFA for 10 min followed by intracellularly staining with rabbit IgG or our antibody.

• WB

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Western blot - Anti-Interferon gamma antibody [EPR28352-7] (AB324874)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23129404; PMID : 11033016; PMID : 35092032; PMID : 26018190)

The identity of the bands higher than 25 kDa are unknown.

In Western blot, Anti-Actin antibody [EPR16875] - Loading Control (ab200658) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-Interferon gamma antibody [EPR28352-7] (ab324874) at 1/1000 dilution

Lane 1:

Untreated mouse spleen cell whole cell lysate at 20 µg

Lane 2:

Mouse spleen cell treated with 50 ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1 μg/ml Ionomycin for 1 hour then add 300ng/ml Brefeldin A for additional 3 hours

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: 17 kDa,42 kDa

false

Exposure time: 15s