Anti-MMP9 antibody [RM1020] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation. 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)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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

• IP

Supplier Data

Immunoprecipitation - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/mmp9-antibody-rm1020-ab283575'>ab283575</a>)

Predicted band size: 78 kDa

Observed band size: 84-92 kDa

false

• WB

Supplier Data

Western blot - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

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 This data was developed using ab283575, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-MMP9 antibody [RM1020] (<a href='/en-us/products/primary-antibodies/mmp9-antibody-rm1020-ab283575'>ab283575</a>) 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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/mmp9-antibody-rm1020-ab283575'>ab283575</a>) 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='/en-us/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

• I-ELISA

Supplier Data

Indirect ELISA - Anti-MMP9 antibody [RM1020] - BSA and Azide free (AB283594)

This data was developed using ab283575, the same antibody clone in a different buffer formulation.

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.