Hearts were extracted at various time points and fixed in 4% paraformaldehyde before paraffin wax embedding. Tissue sections (5 μm) were stained as we described previously with Miller’s elastic van Gieson (MVG) stain (40 (link)) or with antibodies for tenascin-C (Santa Cruz Biotechnology, sc-8694) (38 (link)). Composite tiled images were prepared from individual photomicrographs so that cross-sections through the whole heart could be observed.
Tenascin c
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Tenascin-C is an extracellular matrix glycoprotein that plays a role in cell adhesion and migration. It is involved in various biological processes, including tissue development, wound healing, and tumor progression.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Santa Cruz Biotechnology (3 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Cxcr4 and α sma, by Abcam (2 mentions)
His tag, by Merck Group (2 mentions)
Subcellular protein fractionation kit, by Thermo Fisher Scientific (2 mentions)
Nucleolin, by Santa Cruz Biotechnology (1 mentions)
Collagen 1a1, by Santa Cruz Biotechnology (1 mentions)
Histone h3, by Merck Group (1 mentions)
P jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
X ray film, by Fujifilm (1 mentions)
P c jun ser63, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Sigenome non targeting sirna, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Tgf β1, by Santa Cruz Biotechnology (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
7 protocols using tenascin c
1
Histological Analysis of Cardiac Tissues
2
Immunoblotting Analysis of Cellular Proteins
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For immunoblotting (IB), whole cell lysates were prepared by scraping cells directly in 2x SDS sample buffer. Extracellular matrix was prepared as we previously described [2 (link)]. Proteins were separated by SDS-PAGE and transferred to membranes. Membranes were blocked with 5% non-fat dry milk in TBST buffer then incubated with antibodies against IGFBP-5 (Gropep, Thebarton, SA, Australia), fibronectin, collagen 1A1, GAPDH, tenascin-C, nucleolin (Santa Cruz, Dallas, TX), Histone H3 (Sigma, St. Louis, MO) or tubulin (Epitomics Inc, Burlingame, CA), washed with TBS three times, then incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz, Dallas, TX). Signals were detected by chemiluminescence (Perkin Elmer, Waltham, MA). Images were analyzed using Image J.
3
Protein Expression Analysis Workflow
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Whole cell lysates were prepared by scraping cells directly in 2× sodium dodecyl sulfate (SDS) sample buffer. In parallel experiments, membrane proteins were extracted using the Subcellular Protein Fractionation kit (Pierce Biotechnology, Rockford, IL) following the manufacturer's instructions. Extracellular matrix was prepared as we previously described.4 Proteins were separated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk in Tris‐buffered saline (TBS)/0.05% Tween‐20 (TBST) buffer then incubated with antibodies against human IGFBP‐4, collagen, fibronectin, tenascin‐C, CTGF, GAPDH (Santa Cruz, Dallas, TX), phosphorylated SMAD2 and SMAD3, total SMAD2 and SMAD3, phosphorylated SMAD1/5/9, phosphorylated (p)‐44/42 MAPK, p‐AKT, p‐P38 kinase, p‐SAPK/JUNK, and antibodies targeting the corresponding total proteins (Cell Signaling Tech, Danvers, MA), CXCR4 and α‐SMA (Abcam, Cambridge, MA), His tag (Sigma), or tubulin (Epitomics Inc, Burlingame, CA), washed with TBS three times, and then incubated with horseradish peroxidase‐labeled secondary antibody (Santa Cruz). Signals were detected by chemiluminescence (Perkin Elmer, Waltham, MA, USA) on a FluorChem R system (ProteinSimple, San Jose, CA). Images were analyzed using Image J.
4
Western Blotting of Cellular Proteins
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PVDF membranes (Bio‐Rad) blotted with proteins from whole cell lysates were probed with antibodies against c‐Jun (1:1,000), JNK (1:1,000), p‐c‐Jun (Ser63) (1:500), p‐JNK (Thr183/Tyr185; 1:500, all from Cell Signaling), tenascin C (1:1,000, Santa Cruz), GAPDH (1:10,000, Sigma‐Aldrich), or tubulin (1:10,000, Sigma‐Aldrich). Following primary antibody incubation, membranes were washed and probed with anti‐mouse IgG‐HRP (1:10,000, Leica) or anti‐rabbit IgG‐HRP (1:10,000, Leica) and exposed to X‐ray films (Fujifilm) or imaged in a ChemiDoc imaging system (Bio‐Rad).
5
siRNA Transfection of HUASMCs
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HUASMCs were transfected with short interfering RNA directed against NFAT5 (5′‐CCA GTT CCT ACA ATG ATA A‐3′), tenascin‐C (Santa Cruz Biotechnology) or ACTBL2 (Qiagen). As control, commercially available siGENOME Non‐Targeting siRNA (Thermo Scientific) was applied. For each well of a 6‐well plate, 3 μg of siRNA was diluted in Opti‐MEM I (Invitrogen) together with 3 μL of MATra‐si reagent (IBA) to give a final volume of 200 μL. After mixing and incubating for 20 minutes at ambient temperature, the solution was added onto the cells, which had been cultivated in 2 mL Opti‐MEM I prior to the transfection. Cells were then incubated on a magnetic plate (IBA) at 37°C and 5% CO2. After 15 minutes cells were washed and cultured in normal cell medium for a resting period of 48 hours for ACTBL2 and TNC or 72 hours for NFAT5 knockdown.
6
Protein Extraction and Analysis Workflow
7
Immunofluorescence Microscopy of ECM Proteins
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For fluorescence microscopy studies, cells were plated on eight-well slide glass and coverslips (75 × 25 mm 1, and 18 × 18 mm 0.16, Superior Marienfeld, Germany). The eight-well slide glass was washed with PBS, and cells were fixed with 4% paraformaldehyde in PBS containing 0.1% triton X-100 and 0.1% BSA for 3 min at room temperature (RT). Primary antibodies, incubated with Tenascin-C, Fibronnectin, and TGF-β1 (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted in 1% BSA were kept overnight at 4°C. After several washes with PBS, the samples were incubated with fluorescent-conjugated secondary antibodies (1:500 for FITC-conjugated anti-rabbit, 1:200 for Alexa Fluor 488conjugated anti-rabbit, and 1:200 for green oregon-conjugated anti-mouse immunoglobulins) for 1 hr at RT and embedded in Vectahield with DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were visualized by an Olympus BX-60 microscope with the appropriate filters. A blue signal represents the nuclear DNA staining with DAPI. Finally, the slides were dehydrated and mounted. Image analysis was done using ImageJ software (http:// rsb.info.nih.gov/ij). Representative images were taken with a Spot 4.3 digital camera and software and edited in Adobe Photoshop.
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