Recombinant Anti-MMP9 antibody [EP1254] (ab76003)

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

Exposure time: 3 seconds; 40 seconds.

ab181602 was used as loading control for GAPDH.

Although MMP9 has been studied in brain in some publications, ab76003 was unable to detect signal in normal brain tissue, this may because MMP9 expression level is low in normal brain and would be increased in abnormal conditions like injury (PMID: 31198417).

Running buffer: MOPS.

Conditions: Denatured/reduced.

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab76003 (rabbit-anti MMP9; 1.5 ug/mL) and ab8245 (loading control to GAPDH;  0.1 ug/mL) for 48 hours at 4°C. Before imaging, antibody binding was detected using infrared-labeled goat anti-rabbit (green) and goat anti-mouse (red) at  1:10,000 dilution for 1 hour at room temperature.

Representitive images for skin wound tissue stained for MMP9.

The effect of diabetes on granulation tissue MMP-9 was also studied in a skin excisional wound model. For these studies, the rats were anaesthetized and the dorsum was prepared for wounding. Four full-thickness circular wounds (8mm²) were then created on the dorsum using a biopsy punch as previously described. Wound area was traced daily for determination of wound healing rate (calculated as change in wound area /day) and at day 6 post wounding the animals (n = 5-6/group) were euthanized and the skin containing the wound tissue was excised. Two wounds were snap frozen in liquid N₂ for later measurement of gene expression and the other wounds were divided in half and fixed in formalin (10%) for histological and immunohistological studies or frozen in OCT for immunofluorescence staining.

Results are from control (CON) diabetic (DM) and insulin treated DM (DM+INS) animals.

For full image please see paper.

Blocking and dilution buffer: 5% NFDM/TBST.

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).

Based on our preliminary data, ab76003 detects no or weak band of interest in the untreated cell lines at the dilution of 1/200. Treatment increasing the expression of MMP-9 is recommended when using this antibody.

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Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labeling MMP9 with purified ab76003 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody (inset).

Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labeling MMP9 with unpurified ab76003 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Unpurified ab76003 staining MMP9 in U87-MG cells treated with domoic acid (ab120338), by ICC/IF. Increase of MMP9 expression correlates with increased concentration of domoic acid, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120338 (domoic acid) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with unpurified ab76003 (1/200) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Overlay histogram showing permeabilized A431 (Human epidermoid carcinoma cell line) cells stained with unpurified ab76003 (pink line). 

Negative control antibody (green line) was rabbit IgG. 

Immunocytochemistry/Immunofluorescence analysis of U-2 OS (human osteosarcoma) cells labeling MMP9 with ab76003 at 1/500 (4.3 μg/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/mL) was used as the secondary antibody. Cells were counterstained with ab195889 Anti-Alpha Tubulin antibody [DM1A] (1/200, 2.5 μg/mL) - Microtubule Marker (Alexa Fluor^® 594). DAPI (blue) was used as a nuclear counterstain.

Confocal image showing cytoplasmic staining onU-2 OS cells, the expression increased after treatment with TPA (200 nM) for 24 hours (middle panel).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control with both TPA treated and untreated U-2 OS cells.