Cultured cells (MDA-MB-231 or HUVECs)were lysed in a buffer containing 150 mM KCl, 10 mM Tris pH 7.4, 1% Triton X-100, phasphatase inhibitor and protease inhibitors cocktail (Complete Mini; Roche, Mannheim, Germany). The protein concentration of each cell homogenate was measured by the Bradford’s method [27 (link)]. In total, 30μgm of each protein sample was loaded onto a gel in order to carry out 10% SDS-PAGE, after which the separated proteins were transferred onto a PVDF membrane (Hybond-C; Amersham Biosciences, NJ, USA). Following this procedure, the membrane was blocked with 5% bovine serum albumin and then a Western blot hybridization was performed using a number of different specific primary antibodies. The individual antibodies used for the Western blotting were against p-eNOS (Ser1177) (Cell Signaling, #9571, MA, USA), eNOS (Cell Signaling, # 5880,MA, USA), TrkB (BD, 610101), BDNF (Genetex, GTX62495, CA, USA), VEGFA (Genetex, GTX 102643, CA, USA), MMP2 (Cell Signaling, #13132,MA, USA), MMP9 (Cell Signaling, #13667,MA, USA), MMP13 (Genetex, GTX100665, CA, USA), RhoA (Cell Signaling #2117,MA, USA), p-Rac1/cdc42 Ser71 (Cell Signaling #2461,MA, USA), Rac1/2/3 (Cell Signaling, #2465,MA, USA), cdc42 (Cell Signaling, #2466,MA, USA) and COX-2(Cayman Chem160112, Michigan, USA).
Mmp13
Manufactured by GeneTex
Sourced in United States, China
MMP13 is a lab equipment product that functions as a matrix metalloproteinase. Matrix metalloproteinases are a family of enzymes involved in the breakdown and remodeling of extracellular matrix components.
Automatically generated - may contain errors
Lab products found in correlation
Adamts4, by Abcam (3 mentions)
Bx53 microscope, by Olympus (2 mentions)
Image pro plus version 6, by Media Cybernetics (2 mentions)
Hybond c, by Cytiva (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
Brain derived neurotrophic factor (bdnf), by GeneTex (1 mentions)
Vegfa, by GeneTex (1 mentions)
P rac1 cdc42 ser71, by Cell Signaling Technology (1 mentions)
Rac1 2 3, by Cell Signaling Technology (1 mentions)
P enos ser1177, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Cdc42, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Aggrecan, by GeneTex (1 mentions)
Caspase 3, by Boster Bio (1 mentions)
P jak2, by Boster Bio (1 mentions)
Adamts5, by Bioss Antibodies (1 mentions)
P stat3, by Boster Bio (1 mentions)
Stat3, by Proteintech (1 mentions)
4 protocols using mmp13
1
Western Blot Analysis of Angiogenic Factors
2
Immunohistochemical Analysis of Cartilage Proteins
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Immunohistochemical analyses were performed to detect the expression levels of aggrecan (1: 500; GeneTex, Inc., USA), collagen II (Col-II) (1: 100; II-II6B3 deposited in DSHB by T.F. Linsenmayer), caspase-3 (1: 200; Boster Co., China), matrix metalloproteinase (MMP)-13 (1: 200; GeneTex, Inc., USA), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 (1: 200; Abcam, Inc., USA) in cartilage. Paraffin sections were deparaffinized with xylene and rehydrated with ethanol. After antigen retrieval with 0.05% trypsin and inactivation of endogenous peroxidases with 0.3% H2O2, the sections were incubated at 4°C overnight with the target protein antibody. The remaining experimental procedures were conducted in accordance with the protocols provided with the PV-6000 DAB detection kit and ZLI-9018 DAB kit (both from ZSGBBIO Corp., China), before being counterstained with hematoxylin. Images were captured at 100× magnification using a BX53 microscope (Olympus, Japan) and semiquantitatively analyzed with Image-Pro Plus version 6.0 software (Media Cybernetics, USA). The average optical density intensity of each protein, expressed as the integrated optical density/mm2, was defined as the value of the integrated optical density divided by the cartilage area.
3
Cartilage Protein Expression Analysis
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The expression levels of aggrecan (1: 300; Abbiotec LLC, San Diego, CA, USA), caspase-3 (1: 200; Boster Co., Ltd., Wuhan, China), MMP-13 (1: 200; Gene Tex Inc., USA), and ADAMTS-4 (1: 200; Abcam Inc., USA) in cartilage were analyzed. Specifically, sections were rehydrated with ethanol following deparaffinization with xylene. After antigen retrieval with 0.05% trypsin and inactivation of endogenous peroxidases with 0.3% H2O2, incubation with the target protein was performed overnight at 4°C. The remaining experimental procedures were conducted in accordance with the protocols provided with the PV-6000 DAB detection kit and ZLI-9018 DAB kit (all from ZSGB-BIO Corp., China). Finally, these sections were counter-stained with hematoxylin.
Immunohistochemical images were captured using a BX53 microscope (Olympus, Tokyo, Japan) at 100× magnification and then semiquantitatively analyzed with Image-Pro Plus version 6.0 software (Media Cybernetics, USA). The average optical density intensity of each protein, expressed as IOD/mm2, was defined as the value of the integrated optical density divided by the cartilage area.
Immunohistochemical images were captured using a BX53 microscope (Olympus, Tokyo, Japan) at 100× magnification and then semiquantitatively analyzed with Image-Pro Plus version 6.0 software (Media Cybernetics, USA). The average optical density intensity of each protein, expressed as IOD/mm2, was defined as the value of the integrated optical density divided by the cartilage area.
4
Immunohistochemical Analysis of Cartilage
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Paraffin sections were routinely deparaffinized, rehydrated, and repaired by 0.05% trypsin and inactivation of endogenous peroxidases with hydrogen peroxide (H2O2) at room temperature for ten minutes. Subsequently, incubation with the antibodies was performed overnight at 4°C. The expression levels of collagen-II (Col-II) (1 μg/ml; DSHB Hybridoma Product II-II6B3, from Linsenmayer TF at Tufts University School of Medicine, Boston, Massachusetts, USA), aggrecan (AGG) (2.85 μg/ml; Gene Tex, San Antonio, Texas, USA), caspase-3 (0.667 μg/ml; Boster, Wuhan, China), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 (2 μg/ml; Abcam, Cambridge, UK), ADAMTS-5 (2 μg/ml; Bioss, Beijing, China), matrix metalloproteinase-13 (MMP-13) (1 μg/ml; Gene Tex), JAK2 (2.267 μg/ml; Proteintech, Wuhan, China), STAT3 (1.933 μg/ml; Proteintech), p-JAK2 (2.5 μg/ml; Boster), and p-STAT3 (2.5 μg/ml; Boster) in the cartilage were analyzed. Finally, the colour brown developed with DAB staining (ZSGB-BIO Corporation, Beijing, China). All sections were measured by Image Pro Plus (IPP) version 6.0 software (Media Cybernetics, Rockville, Maryland, USA). Then, the mean intensity of optical density (IOD) was measured as previously described.5,18 (link)
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