Anti-IL-10 antibody [RM1325]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-10 antibody [RM1325] (AB324290)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with Lipopolysaccharides (100ng/ml) for 4 hours add Brefeldin A (1ug/ml) for 3 hours (Lower left and lower right) / Untreated RAW264.7 (Upper left and upper right) cells labelling IL-10 with ab324290 at 1/5000 dilution (0.01ug) / 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-10 antibody [RM1325] (AB324290)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling IL-10 with ab324290 at 1/50 (9.74 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in RAW 264.7 cell line treated with Lipopolysaccharides(100ng/ml) for 4hours add Brefeldin A(1ug/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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ab195889 200 2.5ug/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/ab150081 1000 2ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-IL-10 antibody [RM1325] (AB324290)

IL-10 was immunoprecipitated from 0.35 mg RAW 264.7 treated with 100ng/mL LPS for 4h add 1ug/ml BFA for 3h whole cell lysate with ab324290 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324290 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-IL-10 antibody [RM1325] (ab324290) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS for 4h add 1ug/ml BFA for 3h whole cell lysate

Lane 2:

ab324290 IP in RAW 264.7 treated with 100ng/mL LPS for 4h add 1ug/ml BFA for 3h 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 ab324290 in RAW 264.7 treated with 100ng/mL LPS for 4h add 1ug/ml BFA for 3h 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: 20 kDa

false

Exposure time: 41s

• I-ELISA

Supplier Data

Indirect ELISA - Anti-IL-10 antibody [RM1325] (AB324290)

Indirect ELISA analysis of ab324290 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Mouse IL-10.

Antigen concentration : 1000 ng/ml

• WB

Supplier Data

Western blot - Anti-IL-10 antibody [RM1325] (AB324290)

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

The expression of IL-10 is upregulated in response to LPS and BFA treatment (PMID : 31447835).

The band beneath the target band (14 kDa) is expected to be cleavaged fragment (PMID : 20949050).

Recombinant multiclonal ab324290 showed stronger specificity than polyclonal ab9969.

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

All lanes:

Western blot - Anti-IL-10 antibody [RM1325] (ab324290) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 40 µg

Lane 2:

RAW 264.7 treated with 100ng/ml LPS for 4 hours, 1ug/ml BFA was then added for additional 3 hours, whole cell lysate at 40 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: 20 kDa,36 kDa

false