Sc 10736

Manufactured by Santa Cruz Biotechnology

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Sc-10736 is a lab equipment product. It serves as a core function, but a detailed description is not available while maintaining an unbiased and factual approach.

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9 protocols using sc 10736

Histological and Immunohistochemical Analysis of Rat Brains

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The brains of E20 (for CD31, MMP2 and MMP9), P0 (for hematoxylin and eosin and collagen IV), or P52 (for Luxol Fast Blue, myelin basic protein and SMI-312) rats that had been perfused with 10% neutral buffered formalin were post-fixed 2–3 days in paraformaldehyde. Brains were transferred to 30% sucrose for cryopreservation then frozen in OCT. Cryosections (10 μm) were stained with hematoxylin and eosin (H&E) or Luxol fast blue (LFB) following standard protocols [28 (link)].
Immunohistochemistry was performed as described [20 (link),28 (link)]. Sections were incubated overnight with primary antibodies, including: goat anti-CD31 (PECAM-1) (1:200, sc-1506; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-MMP2 (1:100; sc-10736; Santa Cruz); rabbit anti-MMP9 (1:200; #ab38898; Abcam, Cambridge, MA); rabbit anti-collagen IV (1:400; #ab6586; Abcam); rat anti-myelin basic protein (MBP) (1:200; #ab7349; Abcam); mouse anti-SMI-312 (1:200; #837901; BioLegend, San Diego, CA) at 4°C. After several rinses in PBS, sections were incubated with species-appropriate fluorescent secondary antibodies (Alexa Fluor 488 and 555, Molecular Probes, Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Controls included the omission of primary antibodies.

Carusillo Theriault B., Woo S.K., Karimy J.K., Keledjian K., Stokum J.A., Sarkar A., Coksaygan T., Ivanova S., Gerzanich V, & Simard J.M. (2017). Cerebral microbleeds in a neonatal rat model. PLoS ONE, 12(2), e0171163.

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Protein Extraction and Western Blot Analysis

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The cells were lysed by incubation for 30 min with NP-40 lysis buffer (20 mM Tris, pH 7.5, 140 mM NaCl, 1 mM EDTA) containing 1% (v/v) Nonidet P-40, 5 µM AEBSF, 1.5 nM aprotinin, 10 nM E-64 and 10 nM Leupeptin. Cells were sonicated and centrifuged at 12,000 × g for 10 min at 4°C to remove insoluble debris. Total proteins (30 µg) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane using semidry transfer apparatus (Trans-Blot SD Semi-Dry Transfer Cell; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 15 V for 30 min. Membranes were blocked with 5% skimmed milk and incubated with specific antibodies against MMP-2 (1:1,000 dilution; sc-10736; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and microtubule-associated protein 1 light chain 3 (LC3) (1:1,000 dilution; AP1801a; Abgent, Inc., San Diego, CA, USA) at 4°C overnight. After three washes with Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000 dilution; sc-2030; Santa Cruz Biotechnology, Inc.). Protein bands were identified by the enhanced chemiluminescence detecting system (Pierce; Thermo Fisher Scientific Inc.), according to the manufacturer's protocol. β-actin was used as a loading control in the stripped blot.

Hah Y.S., Kim J.G., Cho H.Y., Park J.S., Heo E.P, & Yoon T.J. (2017). Procyanidins from Vitis vinifera seeds induce apoptotic and autophagic cell death via generation of reactive oxygen species in squamous cell carcinoma cells. Oncology Letters, 14(2), 1925-1932.

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Western Blot Analysis of Protein Targets

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Protein from tissues and cells were isolated using RIPA buffer. The protein levels of targets were detected using western blotting method as described in our previous study.^{33 (link)} Primary antibodies against CARD9 (sc-99054) and MMP2 (sc-10736) were obtained from Santa Cruz Biotechnology; MMP3 (ab52915) and MMP9 (ab38898) from Abcam; cleaved caspase 3 (9661 S), caspase 3 (9662 S), phosphorylated NF-κB p65 (3033 S), and NF-κB p65 (8242 S) from Cell Signaling Technology; GAPDH (TA-08) from ZSGB-Bio. The primary antibodies were all diluted to 1:1000. Infrared fluorescence-labeled secondary antibodies were used with a dilution of 1:10000. The membranes were scanned using an Odyssey Infrared Imaging System (Li-Cor Bioscience, Lincoln, NE, USA).

Liu Y., Shao Y.H., Zhang J.M., Wang Y., Zhou M., Li H.Q., Zhang C.C., Yu P.J., Gao S.J., Wang X.R., Jia L.X., Piao C.M., Du J, & Li Y.L. (2023). Macrophage CARD9 mediates cardiac injury following myocardial infarction through regulation of lipocalin 2 expression. Signal Transduction and Targeted Therapy, 8, 394.

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Immunomodulatory Molecule Detection

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Fetal bovine serum (FBS) and modified Eagle’s medium (MEM) were purchased from Gibco (Grand Island, N.Y, USA), and phosphate-buffered saline (PBS), Western Breeze Chromogenic Kit anti-mouse, anti-rabbit, and anti-goat, and LysoTracker Green DND-26 were bought from Invitrogen (Carlsbad, CA, USA). Kit lactic dehydrogenase-based, sulfanilamide, H₃PO₄, and N-1-naphthylethylenediamine dihydrochloride were obtained from Sigma-Aldrich, St. Louis, MO, USA. Antibodies for IL-1β (H153): sc-7884, IL-2 (C2-1-hIL-2): sc-32295, IL-6 (E-4): sc-28343, IL-8 (807): sc-52870, IL-18 (H-173): sc-7954, tumor necrosis factor-alpha (TNF-α) (N-19): sc-1350, cyclooxygenase-2 (COX-2) (M-19): sc-1747, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) (H-300): sc-13032, matrix metalloproteinase-2 (MMP-2) (H-76): sc-10736, matrix metalloproteinase-9 (MMP-9) (M-17): sc-6841, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 (A): sc-109 and β-actin (C4): sc-47778 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Invitrogen (Carlsbad, CA, USA). All of the other chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO, USA).

Voicu S.N., Balas M., Stan M.S., Trică B., Serban A.I., Stanca L., Hermenean A, & Dinischiotu A. (2019). Amorphous Silica Nanoparticles Obtained by Laser Ablation Induce Inflammatory Response in Human Lung Fibroblasts. Materials, 12(7), 1026.

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Cardiac Tissue Histological and Immunoanalysis

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Heart tissues were fixed, embedded and cut into sections as described previously.^{33 (link)} Subsequently, the cardiac sections were subjected to various staining procedures: hematoxylin and eosin (H&E) staining for morphological observation, Sirius red and Masson’s trichrome staining for fibrosis and infarct area analysis, and wheat germ agglutinin (Sigma-Aldrich) staining for cellular hypertrophy evaluation. For immunohistochemical analysis, sections were labeled with primary antibodies followed by HRP-linked secondary antibodies. Diaminobenzidine tetrahydrochloride was used for the colorimetric reaction. For immunofluorescence, frozen sections were labeled with primary antibodies followed by Alexa Flour 488- or Alexa Flour 555-conjugated secondary antibodies (Invitrogen). Antibodies against Mac3 (sc-19991), MMP2 (sc-10736), and CARD9 (sc-49408) were purchased from Santa Cruz Biotechnology; anti-CD31 (DIA-310) from Dianova; anti-MMP9 (ab38898 and ab283575), neutrophils (ab2557), F4/80 (ab6640), and SLC22A17 (ab124506) from Abcam; anti-α-actinin (A7811) from Sigma-Aldrich; anti-LCN2 (AF1857) from R&D System. Anti-CD31 was diluted to 1:50, and all other primary antibodies were diluted to 1:100. A Nikon ECLIPSE Ni microscope (Nikon, Japan) or a laser confocal microscope (Leica, Buffalo Grove, IL, USA) were used for image capture. ImagePro Plus 3.0 was used for analysis.

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Quantitative Western Blot Analysis

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Whole-cell extracts were prepared by resuspending cell pellets in a RIPA buffer supplemented with a cocktail of protease (Roche). The protein concentrations were quantified by the Bradford method. A total of 30 μg of protein extract was subjected to 10% SDS-PAGE gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), and blotted overnight with rabbit polyclonal anti-MMP-2 antibody (sc-10736, 1:800, Santa Cruz), rabbit polyclonal anti-VEGF antibody (sc-507, 1:350, Santa Cruz) and mouse monoclonal anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (sc-365062, 1:8000, Santa Cruz) in 5% BSA in Tris-buffered saline and 0.01% Tween-20. Peroxidase-conjugated secondary antibodies (sc-2371, 1:5000, Santa Cruz) were used and developed with the chemiluminescence reagent ECL Plus using hyperfilm (Amersham Biosciences). Quantification of the western blots was performed using Quantity One software. Each experiment was conducted three times, and a representative result was shown.

Zhao Y., Wang Q., Zeng Y., Xie Y, & Zhou J. (2022). Gastrin/CCK-B Receptor Signaling Promotes Cell Invasion and Metastasis by Upregulating MMP-2 and VEGF Expression in Gastric Cancer. Journal of Cancer, 13(1), 134-145.

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Western Blotting Pathway Analysis

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Western blotting was performed as described previously [28 (link)]. The protein-transferred membranes were incubated overnight at 4°C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-TrkB (1:500, ab197072, Cambridge, MA, USA), BDNF (1:200, sc-546, Santa Cruz Biotechnology), MMP-2 (1:200, sc-10736, Santa Cruz Biotechnology), MMP-9 (1:200, sc-6840, Santa Cruz Biotechnology), E-cadherin (1:200, sc-7870, Santa Cruz Biotechnology), vimentin (1:200, sc-6260, Santa Cruz Biotechnology), SLUG (1:200, sc-15391, Santa Cruz Biotechnology), SNAIL (1:200, sc-10433, Santa Cruz Biotechnology), twist (1:200, sc-15393, Santa Cruz Biotechnology), VEGF-C (1:200, sc-7133, Santa Cruz Biotechnology), VEGF-D (1:200, sc-7603, Santa Cruz Biotechnology), p-MEK-1/2 (1:200, sc-7995, Santa Cruz Biotechnology), Erk1/2 (1:200, No9102 Cell signaling Technology), p-Akt1/2/3 (1:200, sc-101629, Santa Cruz Biotechnology), or Akt1/2/3 (1:200, sc-8312, Santa Cruz Biotechnology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added and the membranes were further incubated for 1 h at room temperature. The antibodies for α-tubulin (1:1000, Sigma-Aldich, St. Louis, MO, USA) and β-actin (1:200, sc-47778, Santa Cruz Biotechnology) were used as protein loading controls.

Kawamoto M., Onishi H., Ozono K., Yamasaki A., Imaizumi A., Kamakura S., Nakano K., Oda Y., Sumimoto H, & Nakamura M. (2017). Tropomyosin-related kinase B mediated signaling contributes to the induction of malignant phenotype of gallbladder cancer. Oncotarget, 8(22), 36211-36224.

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Propofol Influence on U373 GBM Cells

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The U373 human GBM cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine (Qiagen, Shanghai, China), 100 U/m penicillin (Qiagen) and 100 mg/ml streptomycin (Qiagen) at 37°C in an atmosphere containing 5% CO₂. Propofol was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) for in vitro analysis. An MMP-2 ELISA kit was obtained from R&D Systems Europe, Ltd. (Abingdon, UK). β-actin (1:300, sc-130656) and MMP-2 (1:2,000, sc-10736) polyclonal rabbit antibodies were supplied from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The U373 cells were treated with control or different concentrations of Propofol (1, 5, 10μg/ml) in this study.

XU J., XU W, & ZHU J. (2015). Propofol suppresses proliferation and invasion of glioma cells by upregulating microRNA-218 expression. Molecular Medicine Reports, 12(4), 4815-4820.

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Western Blot Analysis of MMPs

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Equal amounts of denatured protein were subjected to 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), including molecular weight markers (PageRuler, #26617, Fermentas and PageRuler Plus, #266201, Fermentas) to estimate the molecular weight sizes of appearing bands, and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were washed in TBST buffer (TBS containing 0.1% Tween-20), and non-specific binding sites were blocked by immersing the membranes in blocking buffer containing 5% non-fat milk (70166, Sigma) in TBST buffer for 1 h on a shaker at room temperature or overnight at 4 °C. Membranes were first probed with the following primary antibodies: α-MMP2 (1:1000, sc-10736, Santa cruz), α-MMP3 (1:1000, ab52915, Abcam) and α-MMP14 (1:1000, ab3644, Abcam). After the first blotting, an anti-vinculin antibody (1:50000, V9131, Sigma) was used to control for protein loading. Next, a peroxidase-conjugated α-rabbit (111-035-144, Jackson ImmunoResearch) secondary antibody was used. Bound antibodies were visualized with the Pierce Enhanced Chemiluminescence (ECL) Plus Western Blotting Substrate detection system (32132, ThermoFisher) according to the manufacturer’s instructions.

Rabieifar P., Zhuang T., Costa T.D., Zhao M, & Strömblad S. (2019). Normal mammary gland development after MMTV-Cre mediated conditional PAK4 gene depletion. Scientific Reports, 9, 14436.

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