α sma

Manufactured by Vector Laboratories

Sourced in United States

α-SMA (Alpha-Smooth Muscle Actin) is a protein marker used in research applications. It is a key component of the contractile apparatus in smooth muscle cells. α-SMA is widely used in immunohistochemistry and Western blotting to identify and quantify smooth muscle cells.

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Lab products found in correlation

α sma, by Merck Group (3 mentions) α sma, by Abcam (3 mentions) Pan cytokeratin, by Vector Laboratories (2 mentions) Vimentin, by Vector Laboratories (2 mentions) Methyl green, by Vector Laboratories (1 mentions) Antigen retrieval solution, by Vector Laboratories (1 mentions) Alexa 594, by Thermo Fisher Scientific (1 mentions) Immpact dab diluent, by Vector Laboratories (1 mentions) Axiovert s100, by Zeiss (1 mentions) Citrisolv, by Thermo Fisher Scientific (1 mentions) Leica refine detection kit, by Leica Biosystems (1 mentions) Leica refine kit, by Leica camera (1 mentions) F4 80, by Thermo Fisher Scientific (1 mentions) Dab substrate, by Vector Laboratories (1 mentions) Goat anti rat igg, by BD (1 mentions) Anti α sma, by Merck Group (1 mentions) Anti cd31, by BD (1 mentions) Vectastain elite abc kit, by Vector Laboratories (1 mentions) Anti α smooth muscle actin α sma, by Santa Cruz Biotechnology (1 mentions) Anti mac2, by Santa Cruz Biotechnology (1 mentions)

10 protocols using α sma

Vascularization and Proliferation in Endometriosis

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Vascularization of the endometriotic lesions was assessed by immunofluorescence for alpha-smooth muscle actin (α-SMA) (Sigma-Aldrich, St. Louis, USA) (1:200, 30 min at RT) in combination with fluorescein-labeled Griffonia simplicifolia isolectin B4 (IB4) (Vector laboratories, Burlingame, CA) (1:50, O/N at 4 • C) to detect the presence of mature (IB4+/α-SMA+) and immature (IB4+/α-SMA-) vessels, as previously described [33] . Negative controls were incubated without primary antibody. Images were obtained with a Leica DMI 3000B (Leica Microsystems Inc). Cell proliferation was detected by staining for Ki-67, as previously described [36] .

Buigues A., Ferrero H., Martínez J., Pellicer N., Pellicer A, & Gómez R. (2018). Evaluation of PAI-1 in endometriosis using a homologous immunocompetent mouse model. Biology of reproduction, 99(2).

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Immunofluorescent Analysis of Lung Tissue

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Paraffin-embedded lung sections were deparaffinized and rehydrated with Citrisolv (Thermo Fisher Scientific, Waltham, MA) and graded ethanol, treated with antigen retrieval solution (Vector Lab, Burlingame, CA), then incubated with 3% hydrogen peroxide, and blocked with 2.5% horse serum in Tris-buffered saline and Tween 20 (TBST) prior to incubation with primary antibodies. For dual fluorescent staining, sections were incubated overnight with mouse monoclonal antimouse α-smooth muscle actin antibody (α-SMA, 1:100, clone 1A4; Sigma, St. Louis, MO) and rabbit polyclonal von-Willebrand Factor (FVIII, 1:500; Sigma, St. Louis, MO). Next day, sections were treated with Alexa 594 (1:500) and Alexa 488 (1:2000) fluorescent secondary antibodies (Invitrogen, Carlsbad, CA). Slides were counterstained with methyl green (Vector Laboratories, Burlingame, CA) to stain the nuclei. Additional slides stained for α-SMA were incubated with an anti IgG antibody followed by the ABC reagent (Vectastatin kit; Vector Laboratories, Burlingame, CA), developed with ImmPact DAB diluent (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin for morphometric analysis. Tissue sections were examined by light microscopy and photographed on a Zeiss Axiovert S100 (Carl Zeiss LLC, Thornwood, NY) at 100× magnification, and images were analyzed using AxioVision software.

Gupta N., Rashid J., Nozik-Grayck E., McMurtry I.F., Stenmark K.R, & Ahsan F. (2017). Cocktail of Superoxide Dismutase and Fasudil Encapsulated in Targeted Liposomes Slows PAH Progression at a Reduced Dosing Frequency. Molecular pharmaceutics, 14(3), 830-841.

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Immunostaining of Sponge Slices

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Immunostaining of sponge slices was performed using a Leica Staining Robot with robotic antigen retrieval for F4/80 using trypsin solution (0.5 mg/mL in PBS, 10 min, 37 °C), or for αSMA manual antibody retrieval using a pressure cooker under standard conditions (citrate NCL pH 6 buffer); antigen retrieval was not performed for CD31. For F4/80 and αSMA, samples were blocked (5 min) with hydrogen peroxide solution from the Leica Refine Detection Kit (Leica Biosystems, Milton Keynes, UK) followed by serum block (30 min; F4/80, Immpress anti-rat (mouse absorbed) kit (Vector Laboratories, Peterborough, UK); αSMA, Mouse on Mouse Abcam Kit (Cambridge, UK)). Thereafter, samples were incubated with primary antibody (30 min; F4/80 1:300 (eBiosciences, Hatfield, UK); αSMA 1:4000 (Sigma-Aldrich, Dorset, UK)) prior to incubation with polymer (30 min, F4/80 Impress Kit as above; αSMA as above). For CD31 a Leica Refine Kit (as above) was used, which included a hydrogen peroxide block (5 min), primary antibody (120 min, 1:200 (Abcam)) and polymer incubation (15 min). Immunostaining was completed with incubation with 3,3′-diaminobenzidine (10 min) and counterstaining with haematoxylin (5 min), both from the Leica Refine Kit.

Gastaldello A., Livingstone D.E., Abernethie A.J., Tsang N., Walker B.R., Hadoke P.W, & Andrew R. (2017). Safer topical treatment for inflammation using 5α-tetrahydrocorticosterone in mouse models. Biochemical Pharmacology, 129, 73-84.

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Immunohistochemical Analysis of Tumor Vessels

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Frozen sections of the tumors were fixed in acetone and an immunoperoxidase staining was performed by standard immunohistochemistry methods. Briefly: after blocking, samples were incubated ON with anti-CD31 (Pharmingen, San Diego, CA), anti-α-SMA (Sigma, San Louis, Missouri) or isotype-matched control antibodies as indicated. After 30 min of incubation with goat anti rat-IgG (Pharmingen, San Diego, CA) or anti mouse-IgG kit (Vector, Burlingame, California), for CD-31 or α-SMA respectively. Sections were developed using Elite-Vectastain ABC-peroxidase (Vector, Burlingame, CA) for 30 min and DAB substrate (Vector, Burlingame, CA). Slides were counterstained with hematoxylin and mounted. Pictures were taken using an Olympus microscope equipped with a digital camera.

Medina M.V., D´Agostino A., Ma Q., Eroles P., Cavallin L., Chiozzini C., Sapochnik D., Cymeryng C., Hyjek E., Cesarman E., Naipauer J., Mesri E.A, & Coso O.A. (2020). KSHV G-protein coupled receptor vGPCR oncogenic signaling upregulation of Cyclooxygenase-2 expression mediates angiogenesis and tumorigenesis in Kaposi’s sarcoma. PLoS Pathogens, 16(10), e1009006.

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Immunohistological Analysis of Kidney Tissues

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For immunohistological analysis, formalin-fixed paraffin-embedded kidney tissues were deparaffinized, rehydrated, and stained with anti-α-smooth muscle actin (α-SMA) (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) or anti-MAC2 (1:100, Santa Cruz Biotechnology) antibodies overnight and at 4°C. On the second day, the kidney sections were washed and incubated with biotinylated anti-mouse secondary antibody (1:200) for 1 h. Finally, α-SMA and MAC2-positive signals were developed using avidin-biotinylated HRP complex (VECTASTAIN ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) followed by counterstaining with hematoxylin. Kidney tissue slides were mounted, and the immunostained kidney sections were examined at ×400 magnification with a light microscope and analyzed using Nikon NIS Elements Software (Nikon Instruments Inc., Melville, NY, USA). The MAC2- and α-SMA-positive renal section areas were examined by two blinded observers and expressed as the percentage area fraction relative to the total area analyzed.

Goorani S., Hye Khan M.A., Mishra A., El-Meanawy A, & Imig J.D. (2024). Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences. Kidney & blood pressure research, 49(1), 69-80.

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Immunocytochemical Characterization of Cell Types

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Cells were seeded in a 96 well plate at 10,000/well in growth media. After 24 hours, cells were fixed in 10% neutral formalin buffer (NBF) for an additional 24 hours, permeabilized with methanol for 10 minutes at −20°C. For primary incubation with mouse monoclonal antibodies, cells were blocked in PBS containing 3.4% mouse FAB fragment for (Jackson Laboratory cat no. 715 001 003) for 1 hour. For all other antibodies, cells were blocked in PBS containing 3% FBS. Cells were incubated with primary antibodies at a 1: 3 dilution to Fibroblast Specific Protein 1 (Fsp1) (Abcam cat no. ab75550), or at a 1:100 dilution to: pan-cytokeratin (Santa Cruz Biotechnology cat no. sc 8018), α−smooth muscle actin (α-sma) (Abcam cat no. 7187), N-cadherin (Santa Cruz Biotechnology cat no. 7339) or Von Willebrand Factor 8 (VWF8, Millipore cat no. Ab7356). pan-cytokeratin and α-sma were detected with secondary anti-mouse-hrp at 1:500 dilution for 2 hours in PBS/3% FBS and incubation with 3,3’-diaminobenzidine substrate (DAB, Vector Laboratories cat no. SK-4100). FSP1, N-cadherin and VWF8 were detected with secondary anti-rabbit-hrp at a 1:500 dilution and DAB substrate. Cells were counterstained with Mayer’s hematoxylin for 2 minutes. Images were captured using the EVOS FL Auto Imaging system.

Bernard S., Myers M., Fang W.B., Zinda B., Smart C., Lambert D., Zou A., Fan F, & Cheng N. (2018). CXCL1 derived from mammary fibroblasts promotes progression of mammary lesions to invasive carcinoma through CXCR2 dependent mechanisms. Journal of mammary gland biology and neoplasia, 23(4), 249-267.

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Dual Immunostaining for PAX2, Vimentin, and α-SMA

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Indirect immunoperoxidase immunostaining was performed for PAX2 as described above, in combination with vimentin (1:100 Santa Cruz, CA, USA) and alpha-smooth muscle actin (α-SMA, 1:400 Abcam, UK).). Omission of the primary antibody was used as a negative control. vimentin and α-SMA staining were visualized with the Vector SG substrate kit; positive cells are bluish-gray color (Vector). Blocking steps were performed following SG substrate color development. Because α-SMA and PAX2 antibody were developed in rabbits, an anti-rabbit IgG antibody Fab fragment (Jackson ImmunoResearch, West Grove, PA, USA) was used to saturate all the binding sites created during the first set of staining. In addition, peroxidase activity derived from the first set of staining was also blocked using alkaline phosphatase/horseradish peroxidase block (SurModics, MN, USA). Next, a second set of staining was performed for PAX2. A rabbit-on-rodent AP-polymer kit (Biocare Medical, CA, USA) was used for additional blocking and substitutive secondary antibody according to the manufacturer`s protocol. Staining was visualized with the Warp Red chromogen kit, red color (Biocare Medical).

Naito S., Pippin J.W, & Shankland S.J. (2014). The glomerular parietal epithelial cell’s responses are influenced by SM22 alpha levels. BMC Nephrology, 15, 174.

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Evaluation of MK2 Inhibitory Peptide

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An MK2 inhibitory peptide with the sequence YARAAARQARAKALARQLGVAA was synthesized by and purchased from EZBioLab with a purity of 95% or greater as determined by mass spectrometry. The Click-iT EdU kit was obtained from Thermo Fisher Scientific. PDGF-BB and LPA were purchased from MilliporeSigma. Antibodies against PCNA (catalog ab912), Ki67 (catalog ab1580), vimentin (catalog ab28028), α-SMA (catalog ab781), SM22 (catalog ab14106), and osteopontin (catalog ab166709) were purchased from Abcam. CREB (catalog 9104), p-CREB (catalog 9198), and additional vimentin (catalog D21H3, 5741) and α-SMA (catalog 19245) antibodies were purchased from Cell Signaling Technology. RAM11 (catalog M0633) antibodies were purchased from DAKO/Agilent. GAPDH (catalog MAB374) and Fn-EDA (catalog F6140) antibodies were purchased from MilliporeSigma. CD31 (catalog AM50226PU-S) antibodies were purchased from OriGene. For IHC, primary antibodies were used at 1:100 (RAM11), 1:150 (vimentin), 1:500 (α-SMA), or 1:25 (CD31) dilutions, and secondary antibodies (catalog BA-9200) were purchased from Vector Laboratories and used at a 1:2000 dilution. For immunocytochemistry, primary and secondary antibodies were used at a 1:500 dilution.

Tierney J.W., Evans B.C., Cheung-Flynn J., Wang B., Colazo J.M., Polcz M.E., Cook R.S., Brophy C.M, & Duvall C.L. (2021). Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch. JCI Insight, 6(19), e142339.

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Immunohistochemical Analysis of Lung Tissue

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Immunohistochemical staining of paraffin-embedded lung tissue slides was performed using the Mouse on Mouse (M.O.M.™) ImmPRESS™ HRP (Peroxidase) Polymer Kit (Vector Laboratories, Burlingame, USA) according to the manufacturer’s instructions. Anti-transforming growth factor beta (TGF-β) (R&D Systems, Wiesbaden, Germany) and anti-smooth muscle actin (α-SMA) (Merck Millipore, Darmstadt, Germany) were used as mouse primary antibodies. ImmPACT™ VIP Peroxidase (HRP) Substrate Kit (Vector Laboratories) was used to detect TGF-β and α-SMA according to the manufacturer’s instructions. Stained sections were counterstained with hematoxylin.

Wirsdörfer F., de Leve S., Cappuccini F., Eldh T., Meyer A.V., Gau E., Thompson L.F., Chen N.Y., Karmouty-Quintana H., Fischer U., Kasper M., Klein D., Ritchey J.W., Blackburn M.R., Westendorf A.M., Stuschke M, & Jendrossek V. (2016). Extracellular adenosine production by ecto-5′-nucleotidase (CD73) enhances radiation-induced lung fibrosis. Cancer research, 76(10), 3045-3056.

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Characterization of Fibroblast Phenotypes

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5000 fibroblasts were seeded per well in a 96 well plate in DMEM/10% FBS overnight. Cells were fixed in 10% neutral formalin buffer for 24 hours, permeabilized in methanol for 10 minutes at -20°C and blocked in PBS containing 3% FBS for 1 hour. Cells were then incubated with the following antibodies (1:100) for 24 hours in blocking buffer: anti-PDGFR-α (Cell Signaling Technology, cat no.5241), anti-FSP1 (Abcam, cat no. ab75550), α-SMA (Abcam cat no. 7817), anti-VEGFR2 (Santa Cruz Biotechnology, cat no.sc-393163) and anti-Pan-Cytokeratin (Biolegend, cat no.628602). Cells were washed in PBS 3 times and incubated with the following secondary antibodies (1:1000): anti-rabbit-biotinylated (Jackson Laboratories, cat no.111-065) to detect PDGFR-α or anti-mouse-biotinylated (Vector Laboratories, cat no.BA-9200) to detect VEGFR2, α-SMA or Pan-Cytokeratin. Biotinylated antibodies were incubated with streptavidin bound to horseradish peroxidase (HRP) (Vector Laboratories, cat no. SK4100) for 30 minutes. FSP1 was detected using secondary anti-rabbit-HRP (1:500, Avantor, cat no. 10150-732). Protein expression was detected through HRP reaction to 3, 3 -diaminobenzidine substrate (Vector Laboratories, cat no.SK4100).

Fang W.B., Medrano M., Cote P., Portsche M., Rao V., Hong Y., Behbod F., Knapp J.R., Bloomer C., Noel-Macdonnell J, & Cheng N. (2023). Transcriptome analysis reveals differences in cell cycle, growth and migration related genes that distinguish fibroblasts derived from pre-invasive and invasive breast cancer. Frontiers in Oncology, 13, 1130911.

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