Anti-IL-12 p40 antibody [EPR28679-579]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-12 p40 antibody [EPR28679-579] (AB326571)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T cells transfected with a mouse IL12B expression vector containing a GFP-tag® (Middle) 293T cells transfected with an empty expression vector containing a GFP-tag® (Right) cells labelling IL-12 p40 with ab326571 at 1/50 dilution (1ug) / Middle and 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IL-12 p40 antibody [EPR28679-579] (AB326571)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse bone marrow derived dendritic cells induced by 1ug/ml LPS for 24 hours and 200ng/ml BFA for 4 hours Untreated mouse bone marrow derived dendritic cells cells labelling IL-12 p40 with ab326571 at 1/1000 (0.509 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 increased cytoplasmic staining in subsets of mouse bone marrow derived dendritic cells (shown in green) treated with 1ug/ml LPS for 24 hours then add 200 ng/ml BFA for 4 hours, showing weak staining in untreated mouse bone marrow derived dendritic cells. 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).

ab8878 Anti-CD11b Rat monoclonal antibody was used to counterstain tubulin at 1/200 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-12 p40 antibody [EPR28679-579] (AB326571)

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20 permeabilized Mouse bone marrow derived Dendritic cells induced by 1ug/ml LPS for 24 hours and 200ng/ml BFA for 4 hours (Lower left and right) Untreated mouse bone marrow derived Dendritic cells (Upper left and right) cells labelling IL-12 p40 with ab326571 at 1/50 dilution (1ug) / 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® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells were co-stained with anti-CD11c conjugated to BV421 and anti-MHCII conjugated to APC/CY7, then fixed with 4% PFA for 10 min followed by intracellular staining. Gated on viable CD11c positive cells.

• WB

Lab

Western blot - Anti-IL-12 p40 antibody [EPR28679-579] (AB326571)

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

The expression of IL12B is upregulated in response to LPS treatment (PMID : 38416039).

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

All lanes:

Western blot - Anti-IL-12 p40 antibody [EPR28679-579] (ab326571) at 1/1000 dilution

Lane 1:

Untreated mouse spleen cell lysate at 80 µg

Lane 2:

Mouse spleen treated with 1ug/ml LPS and 1ug/ml BFA for 16h cell lysate at 80 µg

Lane 3:

Mouse bone marrow derived Dendritic cell lysate at 80 µg

Lane 4:

Mouse bone marrow derived Dendritic cells induced by 1ug/ml LPS for 24 hours and 200ng/ml BFA for 4 hours cell lysate at 80 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: 40 kDa,36 kDa

false

Exposure time: 125s