The chemical substances, antibodies, and reagents used herein were obtained from various sources. Dulbecco's modified Eagle medium (DMEM), phosphate buffer saline (PBS), Collagenase, fetal bovine serum (FBS), and Lipofectamine 3,000 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); Mitochondrial division inhibitor 1 and MitoSOX Red Mitochondrial Superoxide Indicator were purchased from MedChemExpress(Shanghai); TRIzol reagent and mRNA qRT-PCR Sybr Green Detection Kit were purchased from Invitrogen (USA). An Annexin V-FITC, Propidium Iodide (PI) Detection Kit,m and A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit were purchased from BD Biosciences (New Jersey, USA); The following primary antibodies were used in this experiment: anti-GAPDH (1:100, Cell Signaling Technology, USA), anti-UCP2 (1:5,000, R&D Systems, Inc, USA), anti-MMP9 (1:1,000, R&D Systems, Inc. USA), anti-TGF-beta (1:1,000, Abcam, Cambridge, Britain), and anti-p-DRP1 (1:1,000, Cell Signaling). MTT assay (Beyotime); LDH assay (Beyotime).
Anti mmp 9
Manufactured by R&D Systems
Sourced in Germany, United States
Anti-MMP-9 is a laboratory reagent used for the detection and quantification of the matrix metalloproteinase-9 (MMP-9) protein. MMP-9 is an enzyme involved in the breakdown of extracellular matrix components and is implicated in various physiological and pathological processes. This reagent can be used in various research applications that require the measurement or analysis of MMP-9 levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti mmp 13, by R&D Systems (2 mentions)
Anti fak, by BD (2 mentions)
Anti cxcr4 cxc motiv chemokinreceptor 4, by Abcam (2 mentions)
Anti ki 67, by Dianova (2 mentions)
Anti vimentin, by Santa Cruz Biotechnology (2 mentions)
Anti slug, by Santa Cruz Biotechnology (2 mentions)
Anti β1 integrin, by Merck Group (2 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (2 mentions)
Alginate, by Merck Group (2 mentions)
Collagenase, by Merck Group (1 mentions)
Ldh assay, by Beyotime (1 mentions)
Anti tgf beta, by Abcam (1 mentions)
Dulbecco s modified eagle medium, by Merck Group (1 mentions)
Mitosox red mitochondrial superoxide indicator, by MedChemExpress (1 mentions)
Anti p drp1, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mtt assay, by Beyotime (1 mentions)
12 protocols using anti mmp 9
1
Mitochondrial Dysfunction and Apoptosis in Cells
2
Antibody panel for cell signaling
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Monoclonal anti-phospho-specific-FAK and anti-FAK antibodies were obtained from Becton Dickinson (Heidelberg, Germany). Anti-Sirt1 and anti-CXCR4 (CXC-Motiv-Chemokinreceptor 4) antibodies were purchased from Abcam PLC (Cambridge, UK). Anti-phospho-specific p65 (NF-κB) and anti-phospho-specific p50 (NF-κB) antibodies were obtained from Cell Technology (Beverly, MA, USA). Anti-active caspase 3, anti-MMP-9 and anti-MMP-13 antibodies were obtained from R&D Systems (Heidelberg, Germany). Monoclonal anti-β1-Integrin and anti-β-actin antibodies were purchased from Sigma-Aldrich Chemie (Munich, Germany). Monoclonal anti-β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alkaline phosphatase–linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from EMD Millipore (Schwalbach, Germany). Anti-Ki-67 and secondary antibodies used for fluorescence labeling were obtained from Dianova (Hamburg, Germany). All antibodies were used at concentrations recommended by the manufacturers.
3
Protein Extraction and Western Blot Analysis
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Total protein was prepared from UCB tissues and cells using RIPA buffer, and protein quantification was conducted by a spectrophotometer (Thermo Fisher Scientific). Protein extracts were subjected to 14% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride membranes (Millipore, Bradford, MA, USA). After blocking with 5% nonfat milk, membranes were probed with primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China) for 1 h at room temperature. Primary antibodies were as follows: anti-Nrf2 (R&D Systems, Minneapolis, MN, USA), anti-Ki-67 (Boster), anti-matrix metalloproteinase (MMP)-2 (R&D Systems), anti-MMP-9 (R&D Systems), anti-cleaved caspase 3 (R&D Systems), anti-p-glycoprotein (p-gp; Abcam, Cambridge, MA, USA) and anti-β-actin (Boster). Immunoblots were developed by ECL reagents (Pierce, Rockford, IL, USA) and quantified by the Image J software (National Institutes of Health, NY, USA).
4
Zymography-based MMP9 Detection
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HT-1080 cells were solubilized in the zymography lysis buffer as described above and divided into equal amounts, one of which was immediately added with SDS sample buffer without reducing agent. The anti-MMP9 (R&D systems) antibody or control IgG and protein G agarose (Thermo Fisher Scientific) were added to the remaining replicate samples and incubated at 4 °C for 2 h. The samples were centrifuged, and the supernatants were collected and subjected to a zymography analysis as describe above.
5
Antibodies and Lentiviral Silencing Constructs
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Primary antibodies for NPM/B23 (ab10530), DNA-PKcs (ab70250), phospho-ATR (T1989) (ab227851), gamma γH2AX (S139) (ab26350), and phospho-ATM (S794) (ab119799) were obtained from Abcam (Cambridge, UK). Antibodies for phospho-p53 (Ser15) (#9284), phospho-ATM/ATR substrates (S/T*Q motif) (#9607), and MMP-2 (#40994) were from Cell Signaling (Beverley, MA, USA). Anti-MMP-9 was from R&D Systems (AF909) (Minneapolis, MN, USA). Anti-F4/80 was from Proteintech Group (28463-1-AP) (Wuhan, China). For short hairpin RNA (shRNA)-mediated gene silencing, 3 different constructs were designed and synthesized by Genepharma (Shanghai, China). The sequence showing the highest efficiency (5′-GCACAGACTGTCTTCCTTA -3′) was further cloned into pGLVU6/GFP lentiviral vector. Lentivirus packaging and purification were provided by Genepharma.
6
Immunohistochemical Analysis of Stem Cell Markers
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Monoclonal anti-ALDH1 was obtained from Acris Antibodies GmbH (Herold, Germany). Monoclonal anti-CD133 and anti-CD44 were purchased from Abcam PLC (Cambridge, UK). Anti-β-actin, anti-cyclin-D1, anti-ICAM-1, anti-vimentin, anti-E-cadherin, anti-Slug, anti-TGF-β3, anti-TGF-β3R and anti-p-Smad2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-MMP-1, anti-MMP-9 and Anti-MMP-13 were purchased from R&D Systems, Inc., (Heidelberg, Germany). Anti-phospho-specific p65 (NF-κB) and anti-phospho-specific p50 (NF-κB) were obtained from Cell Technology (Beverly, MA, USA). Neutralizing pan-TGF-β antibody, normal rabbit IgG and anti-β1-Integrin were purchased from Sigma-Aldrich Chemie (Munich, Germany). Anti-Ki-67 and secondary antibodies used for fluorescence labelling were purchased from Dianova (Hamburg, Germany). Alkaline phosphatase linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from Millipore (Schwalbach, Germany).
7
Molecular Mechanisms of Pentobarbital-Induced Necroptosis
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Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).
8
Characterizing Immune Cell Phenotypes
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Human FcR Binding Inhibitor (eBioscience) was added to the samples (1:200) and incubated for 20 min on ice to block non-specific Fc-receptor binding. After, cells were washed with FACS buffer and stained with surface antibodies, which included anti-CD45 (HI30, eBioscience); anti-CD40 (5C3, BioLegend); and anti-CD127 (A019D5, BioLegend), at room temperature for 15 min in the dark. Samples were washed twice with FACS buffer. Intracellular staining with anti-CD68 (eBioY1/82A, eBioscience), anti-arginase-1 (R&D systems), and anti-MMP9 (R&D systems) was performed using the Fixation and Permeabilization Solution kit (BD Biosciences). Dead cells were excluded using 7-AAD staining. Samples were run on BD LSRII flow cytometer and data were analyzed using FACSDiva software and FlowJo.
9
Immunohistochemical Analysis of Thrombus
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Consecutive deparaffenizations of paraffin slices were performed. To minimize non-specific staining, the slices were successively processed with 30% H2O2 to impede peroxidase activity and 1% bovine serum albumin to obstruct the non-specific reaction. Next, tissue sections were separately covered with the following different antibodies at the most appropriate concentration: anti-MPO (1:200, Abcam, Cambridge, UK), anti-F4/80 (1:100, Abcam, Cambridge, UK), anti-MCP1 (1:200, Abcam, Cambridge, UK), anti-MMP9 (1:100, R&D Systems; Minneapolis, United States), and anti-VEGF (1:200, Abcam, Cambridge, UK). Finally, color development was carried out using a DAB kit after the slice had been co-incubated with the polink-2 plus Rpolymer HRP detection system (Zsbio; Beijing, China). Five high power areas (×400) inside the thrombus were recorded blindly, and the total number of positively stained cells was summed.
10
Immunohistochemical Analysis of NGAL and MMP-9
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