Brain tissues were homogenized in RIPA lysis buffer and cleared by centrifugation at 12,000 rpm for 30 min at 4°C. Proteins were separated using SDS-PAGE and transferred onto PVDF membranes. Following blocking, membranes were incubated with either mouse anti-MMP-2 (1:400, Santa Cruz, USA), goat anti-MMP-9 (1:400, Santa Cruz, USA), mouse anti-MT1-MMP (1:400, Santa Cruz, USA), or mouse anti-actin (1:5,000, Abcam, Cambridge, UK) antibodies overnight at 4°C. The next day, membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-goat IgG (1:5,000, Promega, USA) secondary antibodies for 2 h at room temperature, and the positive signal was detected with an enhanced chemiluminescent substrate (Thermo Fisher) on a Bio-Rad ChemiDoc XRS digital documentation system. The relative protein expression was compared among rats of different experimental groups.
Goat anti mmp 9
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-MMP-9 is a primary antibody that recognizes matrix metalloproteinase-9 (MMP-9), an enzyme involved in the breakdown of extracellular matrix. It can be used for the detection and quantification of MMP-9 in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti mt1 mmp, by Santa Cruz Biotechnology (2 mentions)
Mouse anti mmp 2, by Santa Cruz Biotechnology (2 mentions)
Mouse anti monocytes macrophages mac387, by Bio-Rad (2 mentions)
Dab kit, by Vector Laboratories (2 mentions)
Rabbit anti matrix metalloproteinase mmp 2, by GeneTex (2 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti actin, by Abcam (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Biotinylated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Mouse anti pcna, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Rabbit anti cd163, by Bioss Antibodies (1 mentions)
Mouse anti α smooth muscle actin, by Santa Cruz Biotechnology (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Rabbit anti ucp1, by Abcam (1 mentions)
5 protocols using goat anti mmp 9
1
Western Blot Analysis of Matrix Metalloproteinases
2
Immunohistochemical Analysis of Matrix Metalloproteinases
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Endogenous peroxidase was quenched using 3% hydrogen peroxide and nonspecific binding was blocked using 2% bovine serum albumin (BSA) for 1 h. Tissue sections were incubated overnight at 4°C with either goat anti-MMP-9 (1:200, Santa Cruz, USA), mouse anti-MMP-2 (1:200, Santa Cruz, USA), or mouse anti-MT1-MMP (1:400, Santa Cruz, USA) antibodies. To detect cerebral microvascular EC or neural proliferation, sections were incubated with mouse anti-PCNA (1:1400, Cell Signaling Technology) or rabbit anti-vWF (von Willebrand factor; 1:400, Dako, Denmark) for 1 h at 37°C. Subsequently, sections were incubated with biotinylated anti-mouse IgG (1:800, Santa Cruz) or anti-goat IgG (1:800, Santa Cruz) secondary antibodies for 1 h at 37°C followed by the avidin-biotin-peroxidase complex (1:100, Vector Laboratories, USA). Immunoreactivity was detected with diaminobenzidine (Boster Biotech Co., P.R. China).
3
Immunohistochemical Analysis of Aortic Wall
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PFA-fixed tissue sections were rinsed in phosphate-buffered saline (PBS) with 1% Triton-X100 and then incubated in 10% oxalic acid for 1 hour. For antigen activation, 0.1% trypsin in PBS was added to the tissue sections. Endogenous horseradish peroxidase (HRP) in the tissue sections was blocked using 3% aqueous hydrogen peroxide in methanol for 8 minutes. After washing in PBS, the tissue sections were blocked with Blocking One Histo. The sections were incubated with the appropriate primary antibody overnight at 4 °C. The histological results from the aortic wall were assessed after staining using the following antibodies: rabbit anti-matrix metalloproteinase (MMP) 2 (1:100; Thermo Scientific), goat anti-MMP9 (1:100; Santa Cruz Biotechnology, Inc.), rabbit anti-MCP-1 (1:50; Novus Biologicals), mouse anti-monocytes/macrophages (MAC387) (1:50; Bio-Rad Laboratories), mouse anti-α-smooth muscle actin (1:400; Santa Cruz Biotechnology, Inc.), rabbit anti-CD163 (1:100; Bioss Antibodies). On the following day, the sections were rinsed in PBS, and incubated with the appropriate secondary antibody conjugated to HRP. Slides were developed with DAB (Vector Laboratories, Burlingame, CA, USA), dehydrated in ethanol (80%, 90%, and 100%), cleared in xylene, and covered with a lipid-soluble mounting medium and glass cover slips.
4
Immunohistochemical Analysis of MMPs
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The deparaffinized tissue sections were permeabilized with 1％ Triton-X100 in PBS and soaked for 1 h in 10％ oxalic acid for bleaching. For antigen retrieval, 0.1％ trypsin in PBS was added to the tissue sections for 10 min. Then, to block endogenous peroxidase, it was soaked in 3％ hydrogen peroxide in methanol for 8 min. After washing with PBS, the tissue sections were blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 30 min. The tissue sections were incubated with the primary antibody overnight at 4 ℃. The following antibodies were used: rabbit anti-matrix metalloproteinase (MMP) -2 (1:100; GeneTex, Irvine, CA, USA) and goat anti-MMP-9 (1:50; Santa Cruz Biotechnology, Dallas, TX) . The following day, the sections were washed with PBS and incubated with the appropriate secondary antibody conjugated to HRP for 30 min. A DAB kit (Vector Laboratories, Burlingame, CA, USA) was used to detect the target proteins. The area of positive staining in immunohistochemistry was calculated by binarizing the images into black and white images using ImageJ.
5
Immunohistochemical Staining of Adipose Tissue
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The deparaffinized tissue sections were permeabilized with 1％ Triton-X100 in phosphate-buffered saline (PBS) and soaked for 1 hour in 10％ oxalic acid to bleach. For antigen retrieval, 0.1％ trypsin in PBS was added to the tissue sections for 10 minutes. Then, for blocking endogenous peroxidase, it was soaked in 3％ hydrogen peroxide in methanol for 8 minutes. After washing with PBS, the tissue sections were blocked with Blocking One Histo for 30 minutes. The tissue sections with primary antibody were overnighted at 4℃. Antibodies are used for the following proteins: rabbit anti-UCP-1 (1:100; abcam, Tokyo, Japan) , rabbit anti-matrix metalloproteinase (MMP) -2 (1:100; GeneTex, Irvine, CA, USA) , goat anti-MMP-9 (1:50; Santa Cruz Biotechnology, Dallas, TX, USA) , and mouse anti-monocytes/macrophages (MAC387) ( 1:50; Bio-Rad Laboratories, Hercules, CA, USA) . The following day, the sections were washed with PBS and incubated with the appropriate secondary antibody conjugated to HRP for 30 minutes. The DAB kit (Vector Laboratories, Burlingame, CA, USA) was used to detect target proteins.
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