αsma fitc

Manufactured by Merck Group

Sourced in United States

αSMA-FITC is a fluorescently-labeled antibody that binds to alpha-smooth muscle actin (αSMA), a cytoskeletal protein expressed in smooth muscle cells. This product can be used in various cell and tissue analysis techniques, such as flow cytometry and immunofluorescence microscopy, to detect and quantify αSMA-positive cells.

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9 protocols using αsma fitc

Modulation of Myofibroblast Differentiation

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Tumor and matched normal Lin-EpCAM-CD73+CD90+ cells were harvested and seeded in glass slide chambers coated with 0.1% gelatin at 50,000 cells/well. 48 hours after seeding, cells were treated with 50 ng/ml recombinant human Jagged-1 (Peprotech, Rocky Hill, NJ, USA) and/or 10 ng/ml recombinant human TGF-β1 (Invitrogen) for three consecutive days. Media was saved and stored at −80 °C. Following treatment, cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 15 minutes. The permeabilization of cells was carried out with 0.1% Triton X-100 (Sigma Aldrich), followed by lock for 1 hour in 2% BSA (Sigma Aldrich) and stained with αSMA-FITC (Sigma Aldrich), Phalloidin-RITC (Molecular Probes, Eugene, OR, USA) and Hoechst 33342 (Invitrogen) for 2 hours. Samples were imaged using a Zeiss laser scanning microscope 710.

Bichsel C.A., Wang L., Froment L., Berezowska S., Müller S., Dorn P., Marti T.M., Peng R.W., Geiser T., Schmid R.A., Guenat O.T, & Hall S.R. (2017). Increased PD-L1 expression and IL-6 secretion characterize human lung tumor-derived perivascular-like cells that promote vascular leakage in a perfusable microvasculature model. Scientific Reports, 7, 10636.

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Tasquinimod Modulates Stromal Cells In Vitro

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For in vitro studies using Tasquinimod, 20,000 Gli1⁺ tdTom⁺ stromal cells were seeded per well in a 6-well plate, in aMEM supplemented by 20% FCS, 1ng/ml bFGF (Peprotech, #450-33), 5 ng/ml murine EGF (Peprotech #315-09). At 24 and 48 h after seeding, cells were treated with recombinant TGF-β (10ng/ml, Invivogen) or PBS control, followed by Tasquinimod treatment (50uM) or DMSO control. For confocal imaging of in vitro Tasquinimod experiments, Gli1⁺ tdTom⁺ stromal cells (20,000 cells/well in 6-well plate) were cultured on glass coverslips and treated as described above. At 72 h post-seeding, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were stained with mouse primary antibody αSMA-FITC (1:100, Sigma, F3777), counterstained with DAPI (4′,6′-′diamidino-2-phenylindole) and mounted in Prolong Diamond. Images were obtained by confocal microscopy on a Leica SP8 microscope.

Leimkühler N.B., Gleitz H.F., Ronghui L., Snoeren I.A., Fuchs S.N., Nagai J.S., Banjanin B., Lam K.H., Vogl T., Kuppe C., Stalmann U.S., Büsche G., Kreipe H., Gütgemann I., Krebs P., Banz Y., Boor P., Tai E.W., Brümmendorf T.H., Koschmieder S., Crysandt M., Bindels E., Kramann R., Costa I.G, & Schneider R.K. (2021). Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis. Cell Stem Cell, 28(4), 637-652.e8.

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Comprehensive Immunostaining Protocol

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Anti-γ-H2A.X IgG (S139) (no. 9718, rabbit monoclonal, Cell Signaling, 1:250), anti-Ki-67 (no. ab15580, rabbit polyclonal, Abcam, 1:50), HNE (HNEJ-2) (no. MHN-100P, mouse monoclonal, Japan Institute for the Control of Ageing, Japan, 1:50), PCNA (no. ab29, mouse monoclonal, Abcam, 1:1,000), α-sma FITC (no. F3777, Sigma, 1:2,000), fluorescein isothiocyanate FITC (no. P510050-8, Dako, 1:2,000), polyclonal swine anti-rabbit biotinylated (no. E0353, Dako, 1:200), rat anti human CD3 (no. MCA1477, Serotec), goat anti rat biotinylated secondary antibody (no. STAR80B, Serotec, 1:200), 53BP1 (no. NB100-305, Novus Biologicals, 1:200).

Jurk D., Wilson C., Passos J.F., Oakley F., Correia-Melo C., Greaves L., Saretzki G., Fox C., Lawless C., Anderson R., Hewitt G., Pender S.L., Fullard N., Nelson G., Mann J., van de Sluis B., Mann D.A, & von Zglinicki T. (2014). Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nature Communications, 2, 4172.

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Immunohistochemistry of Vascular Markers

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Sections were brought at RT from −80 °C and fixed with 4% PFA (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at RT, followed by 3 washings in PBS 1X, 5 min each. PBS 1X + 0.01% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at RT was used to permeabilize tissues. Sections were then incubated with a blocking solution of 10% donkey serum in PBS 1X, for 1 h at RT. Sections were then incubated overnight at 4 °C with rat anti-mouse CD31 (1:50, Clone MEC 13.3, BD PharMingen, Franklin Lakes, NJ, USA) primary antibody, diluted in blocking solution (1:50). After 3 PBS 1X washes of 5 min, donkey anti-rat secondary antibody Dylight 650 (1:100, SA5-10029, Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) was diluted in PBS 1X and added for 1h at room temperature to sections. After PBS 1X washing, fluorescent conjugated Isolectin-594 (1:100, I21413, Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) and α-SMA Fitc (1:100, F3777, Sigma-Aldrich, St. Louis, MO, USA) antibodies diluted in PBS were incubated for 1h at RT. Nuclei were counterstained with DAPI (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at RT. Slides were mounted with PBS 1X-Glycerol (Sigma-Aldrich, St. Louis, MO, USA) at 1:1 ratio and coverslips. Images were acquired with a Leica DMi8 fluorescence microscope.

Tripodi L., Molinaro D., Farini A., Cadiao G., Villa C, & Torrente Y. (2021). Flavonoids and Omega3 Prevent Muscle and Cardiac Damage in Duchenne Muscular Dystrophy Animal Model. Cells, 10(11), 2917.

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Quantifying Myofibroblast Content Using Immunofluorescence

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Myofibroblasts are histologically defined as α-SMA positive cells, co-expressing fibroblast and vimentin markers. Myofibroblast cellular content was quantified using the percentage of cells expressing all these three markers. To detect myofibroblasts, immunofluorescent labeling with combined primary antibodies against the following antigens were adopted, rabbit polyclonal FSP-1 (ab27957-Abcam, MA, 1:100 dilution), mouse monoclonal α-SMA (α-SMA-FITC, F3777-Sigma Aldrich, MO, 1:500 dilution), and vimentin (ab45939-Abcam, MA, 1:100 dilution). Secondary antibodies, donkey anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 594 (A-21202 and A-21207, respectively; Invitrogen, NY, 1:500 dilution) were used. Mounting medium containing DAPI (H-1200-Vector Lab, CA) was then applied. Quantification of the co-expression of α-SMA with FSP-1, and α-SMA with vimentin (myofibroblast) were performed in a blinded fashion. Cellular co-expression of randomly selected images captured at 20× magnification were quantified. Combined cellular co-expression of α-SMA with FSP-1 and α-SMA with vimentin (myofibroblast) were analyzed and scored as percentage of positive myofibroblast cell content. Images were acquired using the Leica TCS SP5 DMI, inverted confocal laser scanning microscope at Mount Sinai’s Shared Resource Facility and analyzed using Leica LAS AF lite software system.

Krishnan P., Purushothaman K.R., Purushothaman M., Turnbull I.C., Tarricone A., Vasquez M., Jain S., Baber U., Lascano R.A., Kini A.S., Sharma S.K, & Moreno P.R. (2016). Enhanced neointimal fibroblast, myofibroblast content and altered extracellular matrix composition: Implications in the progression of human peripheral artery restenosis. Atherosclerosis, 251, 226-233.

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Quantifying Pulmonary Vascular Remodeling

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Pulmonary vascular remodeling was measured by degree of occlusion of capillary arteries on 7 μm-thick sections of frozen lung tissue. Slices were fixed with acetone for 10 minutes at room temperature and then saturated with human (10%) and donkey (10%) sera in PBS for 1 hour at room temperature. We used mouse anti alpha Smooth Muscle Actin (α-SMA)-FITC from Sigma-Aldrich (clone 1A4, dilution 1/100) and rabbit anti von Willebrand Factor (Dako, dilution 1/100). Primary antibodies were incubated overnight at 4°C. Antibody binding was detected with secondary donkey anti-rabbit-Cy3 (1/100) from Jackson ImmunoResearch. One lung section per rat was analyzed (n = 14 rats per group) and all capillary arteries were classified in 4 categories: not muscularized, partially muscularized, fully muscularized and completely occluded according the presence or not of SMC-actin staining around vWF + precapillary arteries (<50 μm).

Chaumais M.C., Ranchoux B., Montani D., Dorfmüller P., Tu L., Lecerf F., Raymond N., Guignabert C., Price L., Simonneau G., Cohen-Kaminsky S., Humbert M, & Perros F. (2014). N-acetylcysteine improves established monocrotaline-induced pulmonary hypertension in rats. Respiratory Research, 15(1), 65.

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Multimarker Immunohistochemistry for Cardiovascular Tissues

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Anti-CX40 (1:500; Alpha Diagnostics Int. Inc.; Cat: CX40-A), αSMA-FITC (1:300; Sigma; Cat: F3777), anti-ERG (1:500; Abcam; Cat: ab92513), CXCR4 (1:125; BD Pharminogen; Cat: 551852 and 1:300; abcam; Cat: 124824), Anti-PH3 (1:500; Millipore; Cat: 06-570), Anti-Myomesin (1:500; DSHB; B4-C), Anti-WGA (1:100, Invitrogen, Cat: W32466), Anti-ENDOMUCIN (1:300, Invitrogen, Cat: 14-5851-82), Anti-VEGFR2 (1:125, R&D Systems, Cat: AF644). Secondary reagents were Alexa fluor-conjugated antibodies (405, 488, 555, 633) from Life Technologies used at 1:250 dilutions.

Das S., Goldstone A.B., Wang H., Farry J., D’Amato G., Paulsen M.J., Eskandari A., Hironaka C.E., Phansalkar R., Sharma B., Rhee S., Shamskhou E.A., Agalliu D., de Jesus Perez V., Woo Y.J, & Red-Horse K. (2019). A unique collateral artery development program promotes neonatal heart regeneration. Cell, 176(5), 1128-1142.e18.

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Temporal Tendon Repair Evaluation

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Hind paws from Scx-Cre^ERT2,Ai9;Scx^GFP reporter mice were harvested prior to tendon surgeries, as well as at D7, 14, 21, 28, 35, and 42 post-surgery. Additionally, hind paws from Scx-Cre; DTR^F/+ were harvested at D28 post-surgery. All hind paws were fixed in 10% neutral buffered formalin (NBF) at room temperature for 72 hr and were subsequently decalcified in Webb Jee EDTA (pH 7.2–7.4) for 14 days at room temperature, processed, and embedded in paraffin. Five-micron sagittal sections were utilized for analysis. For immunofluorescence staining, Scx-Cre^ERT2,Ai9;Scx^GFP sections were stained with GFP (1:500, Cat#: MA5-15256, INVITROGEN, Waltham, MA), tdTomato (1:500, Cat#: AB8181, SICGEN, Cantanhede, Portugal), and α-SMA-FITC (1:500, Cat#: F3777, Sigma Life Sciences, St. Louis, MO). Scx-Cre; DTR^F/+ repair sections were stained with Postn (1:300, Cat#: AB215199, ABCAM, Cambridge, United Kingdom). All sections were counterstained with the nuclear DAPI stain and imaged with a VS120 Virtual Slide Microscope (Olympus, Waltham, MA). Additionally, Scx-Cre; DTR^F/+ repair sections were stained with Alcian blue/hematoxylin and Orange G (ABHOG) to assess tissue morphology and collagen deposition. Finally, Scx-Cre; DTR^F/+ repair sections were further imaged using Second Harmonic Generation (SHG) imaging to facilitate collagen organization and deposition.

Korcari A., Muscat S., McGinn E., Buckley M.R, & Loiselle A.E. (2022). Depletion of Scleraxis-lineage cells during tendon healing transiently impairs multi-scale restoration of tendon structure during early healing. PLoS ONE, 17(10), e0274227.

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Evaluating Vasoactive Pharmacological Agents

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The following drugs and doses were used: angiotensin II (Merck Millipore), 10 μg/kg for acute experiments, 1 mg/kg/day in minipumps, 10 -10 to 10 -5 M for myography; ATR1 antagonist (candesartan, Astra Zeneca), 10 mg/ml in drinking water; ACh (Sigma) 10 μg/kg in vivo, 10 -10 to 10 -5 M for myography, 5 µM for western blot analysis; SNP (Sigma) 200 μg/kg in vivo, 10 -10 to 10 -5 M for myography, 5 µM for western blot analysis; apocynin (Cayman Chemical) 1.5 mmol/L, added daily to the drinking water; troglitazone (Cayman Chemical) 10 μg/kg/day i.p. or 50 μg/kg/day oral gavage; GW9662 (Cayman Chemicals) 10 μg/kg/day i.p.; GW1229 (Adipogen) 10 μg/kg/day i.p. or 50 μg/kg/day oral gavage; GW0742 (Tocris) 5 μg/kg/day i.p. or 20 μg/kg/day oral gavage; lucigenin (Cayman Chemical) 5 µM. The following antibodies were purchased from Cell Signaling Technology: eNOS (rabbit polyclonal, 9572), phosphoeNOS (Ser1177, rabbit polyclonal, 9571), VASP (9A2, rabbit monoclonal antibody); other antibodies were phosphoVASP-biotinylated (Ser239, 16C2, mouse monoclonal, Enzo Life Sciences), RGS5 (chicken polyclonal, XW-7534, ProSci Inc.), αSMA-FITC (1A4, mouse monoclonal, Sigma), CD31 (rabbit polyclonal, ab124432, Abcam).

Holobotovskyy V., Chong Y.S., Burchell J., He B., Phillips M., Leader L., Murphy T.V., Sandow S.L., McKitrick D.J., Charles A.K., Tare M., Arnolda L.F, & Ganss R. (2015). Regulator of G protein signaling 5 is a determinant of gestational hypertension and preeclampsia. Science translational medicine, 7(290).

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