Anti-CXCL2 antibody [EPR28746-89]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Immunohistochemical analysis of paraffin-embedded (A) Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with lipopolysaccharide (LPS, 100 ng/mL) and Brefeldin A (300 ng/mL) for 4 hours. (B) Untreated Raw 264.7. tissue labeling CXCL2 with ab317569 at 1/500 (0.986 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in (A) Raw 264.7 treated with lipopolysaccharide (LPS, 100 ng/mL) and Brefeldin A (300ng /mL) for 4 hours. No staining in (B) untreated Raw 264.7.
The section was incubated with ab317569 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Immunohistochemical analysis of paraffin-embedded (A) Mouse colon of Crohn's disease model. (B) Mouse normal colon. tissue labeling CXCL2 with ab317569 at 1/500 (0.986 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse colon of Crohn's disease model, no staining in mouse normal colon.
The section was incubated with ab317569 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling CXCL2 with ab317569 at 1/100 (4.93 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 cytoplasmic staining in Raw 264.7 cells (shown in green) treated with lipopolysaccharide (LPS, 100 ng/ml) and Brefeldin A (300ng/ml) for 4 hours. The counterstain was observed in magenta. Nuclear DNA was labeled 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 1/200 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 1/1000 2 ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Immunohistochemical analysis of paraffin-embedded (A) Mouse lung treated with lipopolysaccharide (LPS, 1 ug/mL) and Brefeldin A (1 ug/mL) for 16 hours. (B) Untreated mouse lung. tissue labeling CXCL2 with ab317569 at 1/500 (0.986 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse lung treated lipopolysaccharide (LPS, 1 ug/mL) and Brefeldin A (1 ug/mL) for 16 hours. Nearly no staining in (B) untreated mouse lung.
The section was incubated with ab317569 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)

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Flow Cytometry (Intracellular) - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS and 300ng/mL BFA for 4h (Red) / Untreated control (Dotted red) cells labelling CXCL2 with ab317569 at 1/500 dilution (0.1ug)/Red (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.

• IP

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Immunoprecipitation - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

CXCL2 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate with ab317569 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317569 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate
Lane 2 : ab317569 IP in RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317569 in RAW 264.7 treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate

All lanes:

Immunoprecipitation - Anti-CXCL2 antibody [EPR28746-89] (ab317569) at 1/30 dilution

All lanes:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate at 3 µg

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

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Western blot - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

The expression of CXCL2 is upregulated in response to LPS treatment (PMID : 31852751).

The identity of the bands between 37 kDa and 75 kDa are unknown.

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

All lanes:

Western blot - Anti-CXCL2 antibody [EPR28746-89] (ab317569) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 80 µg

Lane 2:

RAW 264.7 treated with 100ng/mL LPS and 300ng/ml BFA for 4h whole cell lysate at 80 µg

Lane 3:

Untreated J774A.1 (mouse reticum cell sarcoma monocyte/macrophage ) whole cell lysate at 80 µg

Lane 4:

J774A.1 treated with 100ng/mL LPS for 3h, 300ng/ml BFA was then added for additional 3h whole 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: 11 kDa,36 kDa

false

Exposure time: 180s

• Dot

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Dot Blot - Anti-CXCL2 antibody [EPR28746-89] (AB317569)

Dot blot analysis of CXCL2 using ab317569 at 1 : 1000 (0.493 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

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

This antibody does not cross-react with mouse CXCL1 and CLCX3.

In dot blot, anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In dot blot, anti-GST antibody [EPR4236] (ab111947) (1 : 1000) staining at 1/1000 dilution.

All lanes:

Dot Blot - Anti-CXCL2 antibody [EPR28746-89] (ab317569) at 1/1000 dilution

Lane 1:

His-tagged mouse CXCL1 recombinant protein

Lane 2:

No tagged mouse CXCL2 recombinant protein

Lane 3:

His/GST-tagged mouse CXCL3 recombinant protein

Secondary

All lanes:

Dot 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

false