The following were stained on paraffin embedded tissue sections (thickness 5 μm) for: CCR2, (1:200; Abcam, Cambridge, MA), FSP-1 (1:500; Millipore, Temcula, CA), Smooth Muscle Myosin Heavy Chain (SMMHC, 1:50; Abcam) and Tlr9 (InvivoGen, San Diego, CA). For CCR2+, FSP-1, SMMHC cell counts, positive cells were counted and totaled in 5 high power fields (hpf, 1000X) radially around the IVC wall or thrombus.15 (link), 25 (link) Trichrome staining in the human sections was done as described.22 (link) Vein wall collagen content was determined using our previously described Sirius Red method.18 (link), 27 (link)To visualize NET in thrombus sections, we stained for cit-H3 (1:500, Abcam) and co-stained with extracellular DNA, labeled with 1uM SYTOX (Invitrogen, Grand Island, NY) at room temperature. Slides were cover slipped with ProLong Gold with DAPI mounting medium (Invitrogen). Pictures were taken using a Nikon Eclipse E400 microscope 1000x equipped with a Nikon Digital Sight DS-U3 camera using the DAPI (nuclei), FITC (SYTOX) and Texas Red (Cit-H3) channels.
Fsp 1
Manufactured by Abcam
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FSP-1 is a laboratory equipment product. It has a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
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Lab products found in correlation
α sma, by Abcam (16 mentions)
Vimentin, by Abcam (7 mentions)
Vimentin, by Santa Cruz Biotechnology (4 mentions)
Fibronectin, by Abcam (3 mentions)
E cadherin, by Abcam (3 mentions)
β actin, by Proteintech (3 mentions)
Pdgfrβ, by Abcam (3 mentions)
α sma, by Merck Group (3 mentions)
F4 80, by Bio-Rad (3 mentions)
E cadherin, by Bioworld Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Epcam, by Abcam (2 mentions)
Collagen 3, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
E cadherin, by BD (2 mentions)
Hif 1α, by Cell Signaling Technology (2 mentions)
α sma, by Proteintech (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
β catenin, by Abcam (2 mentions)
47 protocols using fsp 1
1
Immunohistochemical Profiling of Venous Thrombosis
2
Western Blot Analysis of Cell Signaling
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The dissected cortical tissues and cultured cells were homogenized in cold RIPA lysis buffer. Nuclear extracts from the cortex were obtained using nuclear and cytoplasmic protein extraction Kit (Beyotime, Shanghai, China). Equal amounts of proteins (50 μg) were loaded into SDS-PAGE (8, 10, or 12%), electrophoresed, and transferred onto PVDF membranes (Millipore, Temecula, CA). Nonspecific binding sites were blocked by incubating with 5% non-fat milk, and then the membranes were incubated with primary antibodies. Antibodies against α-SMA (1:1000; Abcam), FSP1 (1:800; Abcam), E-cadherin (1:800; Bioworld), Vimentin (1:4000; Abcam), IKKβ (1:1000; Abcam), IκBα (1:1000; Abcam), P65 (1:5000; Abcam), p-IκBα (1:2000; RabMAb), H3 (1:2000; RabMAb), and GAPDH (1:5000; Abcam). Immunoreactive bands were visualized with the enhanced chemiluminescence (ECL) chemiluminescence system (Millipore). GAPDH or H3 was used as loading control for cytoplasmic or nuclear proteins. The bands were semi-quantified using ImageJ software. All data represent at least three experiments and done in triplicate (mean ± SD).
3
Protein Expression Analysis in Mouse Hearts
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Proteins were extracted from dissected mouse hearts or cultured cells, and equal amounts of protein were separated using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene fluoride membranes. The membranes were blocked for 2 hrs with 5% non‐fat milk, washed for 5 min. in Tris‐buffered saline containing 0.1% Tween‐20 (TBS‐T) and then incubated overnight at 4°C with primary antibodies; the antibodies used were against collagens I and III, VE‐cadherin, CD31, α‐smooth muscle actin (α‐SMA), vimentin, fibroblast‐specific protein (FSP)‐1, MMP‐2 and MMP‐9 (Abcam, Cambridge, MA, USA); and p38 MAPK, phosphor (p)‐p38 MAPK, ERK1/2 and p‐ERK1/2 (Cell Signaling Technology, Beverly, MA, USA). The membranes were washed thrice for 10 min. with TBS‐T, incubated with horseradish peroxidase‐conjugated secondary antibodies (1:10,000, 1 hr, room temperature), washed thrice more for 15 min. with TBS‐T and then incubated with enhanced chemiluminescence substrate to visualize the immune‐reactive protein bands.
4
Comprehensive Immunohistochemical Analysis of Tissue Samples
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The following antibodies were used: N-cadherin (clone GC4, Sigma-Aldrich C3865, 1:100 dilution), FSP1 (Abcam ab58597, 1:100 dilution), PDGFRα (R&D Systems AF1062, 1:50 dilution), Collagen I (Rockland 600–401–103–0.1, 1:150 dilution), Collagen III (Abcam ab7778, 1:150 dilution), Fibronectin (Abcam ab23750, 1:200 dilution), α-SMA (Abcam ab5694, 1:100 dilution), CD31 (Novus Biologicals NB100-2284, 1:500 dilution), EpCAM (Abcam ab92382, 1:500 dilution), Lyve-1 (Abcam ab14917, 1:500 dilution), Myf-5 (clone C-20, Santa Cruz sc-302, 1:500 dilution), CD45 (clone IBL-3/16, Abcam ab23910, 1:500 dilution), FABP4 (clone EPR3579, Abcam ab92501, 1:500 dilution), F4/80 (Abcam ab90247, 1:500 dilution), Cytokeratin 14 (clone EPR17350, Abcam ab181595, 1:200 dilution), Integrin αv (clone EPR16800, Abcam ab179475, 1:500 dilution), α1-catenin (clone EP1793Y, Abcam ab51032, 1:500 dilution), decorin (Abcam ab175404, 1:500 dilution), DLK-1 (Abcam ab21682, 1:200 dilution) and Ki67 (clone SP6, Abcam ab16667, 1:500 dilution). Fluorophore-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (1:500 dilution). Exherin (ADH-1) was from TargetMol (T2637).
5
Western Blotting Analysis of EMT Markers
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All the Western blotting analyses were performed as described previously (Zhao et al., 2015 (link); Chen et al., 2017a (link),c (link); Wang et al., 2017a (link)). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride (PVDF) membranes (10600023, AmershamTM HybondTM, GE Healthcare, United States) by electroblotting. After blocking in Tween-20 buffer and non-fat milk blocking buffer, the membranes were incubated at 4°C over-night with primary antibodies against proteins including E-cadherin (1:500, Abcam, United States), FSP1 (1:1000, Abcam, United States), vimentin (1:200, Abcam, United States), and collagen III (1:5000, Abcam, United States). Membranes were washed and incubated with appropriate horseradish peroxidase-conjugated goat anti-mouse (1:5000, Abbkine, United States) and goat anti-rabbit (1:5000, Abcam, United States) secondary antibodies for 1 h. Blots were detected by enhanced chemiluminescence (RPN2232, GE Healthcare, United States) and band density was quantified by Image J 1.48v software. GAPDH served as the internal control.
6
Western Blotting and qPCR Analysis
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Western blotting and quantitative PCR (RT‐qPCR) were performed following the previous report.
43 (link) Following antibodies were used in Western blotting: Actin, FSP1, ACSL4, SLC7A11, VDAC2, and GPX4 rabbit polyclonal antibodies (1:1000; Abcam); VCAM‐1 and ICAM‐1 (1:500; Santa Cruz Biotechnology, Inc, Dallas, TX, USA). The primers used in RT‐qPCR are shown in TableS1 .
43 (link) Following antibodies were used in Western blotting: Actin, FSP1, ACSL4, SLC7A11, VDAC2, and GPX4 rabbit polyclonal antibodies (1:1000; Abcam); VCAM‐1 and ICAM‐1 (1:500; Santa Cruz Biotechnology, Inc, Dallas, TX, USA). The primers used in RT‐qPCR are shown in Table
7
Antibody and Cell Purification Protocol
8
Immunohistochemical Analysis of Tissue Markers
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Sections of 5 μm in thickness paraffin block were placed on adhesive-coated slides. Then, sections were deparaffinized with xylene, rehydrated in graded ethanol (from 100 to 70%) and heated for 30 min in a sodium citrate buffer to increase epitope exposure. Additionally, slides were treated with 0.3% (v/v) H2O2 in for 5 min, washed with 0.01 M PBS and blocked with 1% BSA, 0.2% Tween 20 in PBS for 1 h at room temperature. Slides were then incubated overnight at 4°C with monoclonal antibody against: Forkhead box P3 (Foxp3 CellSignaling, Danvers, MA, United States), monoclonal antibody against fibroblast surface protein 1, FSP-1 (Abcam, Cambridge, United Kingdom), monoclonal antibody against alpha-smooth muscle actin, α-SMA (Abcam, Cambridge, United Kingdom), and monoclonal antibodies against E-cadherin and N-cadherin (CellSignaling, Danvers, MA, United States). The optimal dilution for all antibodies was 1:100. The reaction antigen-antibody was visualized with avidin-biotin-peroxidase by using 3,3-diaminobenzidine as the chromogen which resulted in brown staining. Slides were counterstained in Harris hematoxylin. Samples were then dehydrated, preserved with a coverslip and reviewed using light microscopy. Pictures were viewed using Olympus FLUOVIEW Viewer software (Tokyo, Japan).
9
Immunofluorescence Staining of Cytoskeletal Markers
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Cells were fixed with 4% PFA for 20 minutes, permeabilized with 1% Triton X‐100 for 15 minutes, and then incubated with goat serum for 1 hour. Subsequently, the cells were incubated with antibodies against cytokeratin (CK, 1:200; Abcam), Vimentin (1:200; Santa Cruz Biotechnology), α‐SMA (1:200; Abcam), FSP‐1 (1:250; Abcam) and FAP (1:250; Abcam) at 4°C overnight. After washing with PBS, cells were incubated with secondary antibodies (1:50) in the dark for 1 hour at 37°C. Then, cell nuclei were stained with DAPI (1:1000; Beyotime, Shanghai, China) for 1 minute. Immunofluorescence was visualized using a Zeiss LSM‐710 laser‐scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
10
Immunostaining of Rat CAECs
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Rat CAECs were fixed in 4% paraformaldehyde and permeabilized with 0.3% Trition-X100. After blocking with 10% BSA for 1 h, normal endothelial cells and TGF-β1-treated endothelial cells were stained for CD31 (Abcam, Cambridge, UK) and FSP-1 (Abcam, Cambridge, UK). And then endothelial cells were incubated with AlexaFluor-conjugated secondary antibodies (Invitrogen Technology, USA) at room temperature in the dark for 1 h. Cells were visualized under a scanning confocal microscope (LSM 510 META, Carl Zeiss, Germany; TCS SP5, Leica, Germany).
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