Anti-MMP9 抗体 [RM1020]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue labelling MMP9 with ab283575 at 1 : 5000 dilution (0.123 μg/ml), followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on immune cells of human gastric carcinoma is observed. The section was incubated with ab283575 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counter stained with hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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-MMP9 antibody [RM1020] (AB283575)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-2 OS (Human bone osteosarcoma epithelial cell) treated with TPA (200nM 24h) (Red) / Untreated control (Green) cells labelling MMP9 with ab283575 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human spleen. The section was incubated with ab283575 for 10 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 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-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the human tonsil. The section was incubated with ab283575 for 10 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [RM1020] (AB283575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-2 OS cells labelling MMP9 with ab283575 at 1/50 (12.26 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). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing increased cytoplasmic staining in U-2 OS cells treated with 12-O-Tetradecanoylphorbol-13-acetate (200nM) for 24hours.

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MMP9 antibody [RM1020] (AB283575)

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 for 4h then together with 1ug/ml BFA for another 3h (Right) / Untreated control (Left) cells labelling MMP9 with ab283575 at 1/500 dilution (0.1ug). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the rat spleen. The section was incubated with ab283575 for 10 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat lung (fresh) tissue labeling MMP9 with ab283575 at 1/100 (6.13 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on macrophages of rat lung is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling MMP9 with ab283575 at 1/5000 (0.123 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the mouse spleen. The section was incubated with ab283575 for 10 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [RM1020] (AB283575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling MMP9 with ab283575 at 1/50 (12.26 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). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Confocal image showing increased cytoplasmic staining in RAW 264.7 cells treated with lipopolysaccharide (100 ng/ml) for 4 hours then together with Brefeldin A (1 ug/ml) for 3 hours.

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] (AB283575)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse lung (fresh) tissue labeling MMP9 with ab283575 at 1/100 (6.13 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on macrophages of mouse lung is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.

• IP

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Immunoprecipitation - Anti-MMP9 antibody [RM1020] (AB283575)

MMP9 was immunoprecipitated from 0.35 mg Mouse lung lysate 10 ug with ab283575 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283575 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse lung lysate 10 ug

Lane 2 : ab283575 IP in Mouse lung lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283575 in Mouse lung lysate

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

Exposure time : 3 min

All lanes:

Immunoprecipitation - Anti-MMP9 antibody [RM1020] (ab283575)

Predicted band size: 78 kDa

Observed band size: 84-92 kDa

false

• WB

Lab

Western blot - Anti-MMP9 antibody [RM1020] (AB283575)

Western blot : Rabbit Recombinant Multiclonal[RM1020] to MMP9 283575 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 90 kDa in Wild-type A549 TPA-treated (80nM, 24h) cell lysates with no signal observed at this size in MMP9 knockout A549 TPA-treated (80nM, 24h) cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MMP9 antibody [RM1020] (ab283575) at 1/1000 dilution

Lane 1:

Wild-type A549 TPA-treated (80nM, 24h) at 20 µg

Lane 2:

Wild-type A549 Vehicle Control TPA (0nM, 24h) at 20 µg

Lanes 3 - 4:

Western blot - Human MMP9 knockout A549 cell line (ab286527) at 20 µg

Lane 5:

Human Lung at 20 µg

Lane 6:

MOLT-4 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 90 kDa

false

• WB

Supplier Data

Western blot - Anti-MMP9 antibody [RM1020] (AB283575)

The expression of MMP-9 can be stimulated by various agents, such as inflammatory cytokine, growth factor, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (PMID : 21047770, 28969043). Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-MMP9 antibody [RM1020] (ab283575) at 1/1000 dilution

Lane 1:

LoVo (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

A549 (Human lung carcinoma epithelial cell) serum starved overnight whole cell lysate at 20 µg

Lane 7:

A549 (Human lung carcinoma epithelial cell) serum starved overnight, then treated with 100ng/ml TPA for 24 hours, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 60s

• I-ELISA

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Indirect ELISA - Anti-MMP9 antibody [RM1020] (AB283575)

ELISA using ab283575 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

• WB

Lab

Western blot - Anti-MMP9 antibody [RM1020] (AB283575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST MMP9 is a glycoprotein (PMID : 29329315). The 92 kDa band likely represents the zymogen prior to activating cleavage of the pro-peptide (PMID : 7688350; PMID : 12901881). Exposure time : Lane 3-4 : 3min ; Others : 37 seconds

All lanes:

Western blot - Anti-MMP9 antibody [RM1020] (ab283575) at 1/1000 dilution

Lane 1:

U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

U-2 OS treated with 200nM TPA for 24h whole cell lysate at 20 µg

Lane 3:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg

Lane 5:

NR8383 (Rattus norvegicus lung macrophage (alveolar)) whole cell lysate at 20 µg

Lane 6:

NR8383 treated with 100ng/ml lipopolysaccharide (LPS) for 4h, then together with 1ug/ml BFA for another 3h whole cell lysate at 20 µg

Lane 7:

Human lung lysate at 20 µg

Lane 8:

Mouse lung lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 78 kDa

Observed band size: 84-92 kDa

false