Desmin

Manufactured by Abcam

Sourced in United States, United Kingdom, Germany

Desmin is a type III intermediate filament protein that is primarily found in skeletal, cardiac, and smooth muscle cells. It plays a structural role in maintaining the integrity and organization of the cytoskeleton within these cell types.

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Lab products found in correlation

α sma, by Abcam (12 mentions) Myogenin, by Abcam (5 mentions) Erk1 2, by Cell Signaling Technology (4 mentions) Podocin, by Abcam (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions) β actin, by Abcam (3 mentions) Myosin, by Abcam (3 mentions) P erk1 2, by Cell Signaling Technology (3 mentions) Vimentin, by Abcam (3 mentions) Tnf α, by Abcam (3 mentions) Proliferating cell nuclear antigen (pcna), by Abcam (3 mentions) α sma, by Cell Signaling Technology (3 mentions) Laminin, by Merck Group (3 mentions) Sm mhc, by Abcam (3 mentions) Collagen 1, by Abcam (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Axio observer a1, by Zeiss (3 mentions) Neurofilament, by Merck Group (2 mentions) Acetylcholine receptor, by Merck Group (2 mentions)

78 protocols using desmin

Muscle Fiber Characterization in Mice

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Mouse muscle samples were frozen in liquid nitrogen‐cooled isopentane in Tissue‐Tek OCT. 10 mm sections were stained with haematoxylin–eosin staining and using the following primary antibodies: Slo1 (1:500, Alomone Labs), WGA (conjugated Alexa Fluor 488, Thermo Scientific, cat. no. W6748), and Desmin (1:1000, Abcam, cat. no. ab32362).
Primary mouse myoblasts were cultured in differentiation medium for 3 days and fixed with 4% paraformaldehyde for 15 min and stained with MYH3 (1:100, Santa Cruz Biotechnology) for myotubes.
For signal detection, Alexa Fluor 594‐conjugated secondary antibodies (1:500, Biolegend, cat. no. 406709) were used. Nuclei were labelled with DAPI.

Xia C., Wang Y., Jiang T., Hu Y., Chen Y., Ma X., Zhang X, & Gao Y. (2023). Slo1 deficiency impaired skeletal muscle regeneration and slow‐twitch fibre formation. Journal of Cachexia, Sarcopenia and Muscle, 14(4), 1737-1752.

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Nerve Crush Injury Protein Analysis

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The middle part of the gastrocnemius muscle and the distal end of crush nerves were harvested and proteins were extracted. Proteins (50 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to blotting membranes. After blocking with nonfat milk, the membranes were incubated with antibodies: S-100 (1:1000 dilution, Merck Millipore, Burlington, MA, USA), neurofilament (1:1000 dilution, Merck Millipore, Burlington, MA, USA), CD 68 (1:1000 dilution, Bio-rad, Hercules, CA, USA), von Willebrand factor (1:200 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), Isolectin B4 (1:200 dilution, Vector Laboratories, Burlingame, CA, USA), desmin (1:1000 dilution, Abcam, Cambridge, MA,USA), acetylcholine receptor (1:1000 dilution, Merckmillipore, Burlington, MA, USA), GAPDH (1: 2000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody and developed using ECL western blotting reagents. The intensity of protein bands was determined by a computer image analysis system (IS1000) (Alpha Innotech Corporation, San Leandro, CA, USA) [29 (link)].

Pan H.C., Chang M.H., Sheu M.L., Chen C.J, & Sheehan J. (2019). Increased angiogenesis by the rotational muscle flap is crucial for nerve regeneration. PLoS ONE, 14(6), e0217402.

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Immunofluorescence Staining of Stem Cells

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Cells were were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, and then incubated with blocking solution (PBS + 1% BSA) at 4°C for 30 min followed by primary antibodies at 4°C overnight. Primary antibodies were diluted in Immnol Fluorence Staining Primary Antibody Dilution Solution (Beyotime, P0108) at the followings ratios: OCT4 (rabbit polyclonal antibody, 1:500, Abcam), SOX2 (rabbit polyclonal antibody, 1:500, Abcam), NANOG (rabbit polyclonal antibody, 1:500, Abcam), SSEA4 (rabbit polyclonal antibody, 1:500, Abcam), CD105 (rabbit monoclonal antibody, 1:500, Abcam), β-Ⅲ-tubulin (rabbit monoclonal antibody, 1:500, Chemicon), AFP (mouse monoclonal antibody, 1:500, Chemicon), Desmin (mouse polyclonal antibody, 1:500, Abcam), Nestin (rabbit monoclonal antibody, 1:200, Chemicon), PDX1 (rabbit monoclonal antibody, 1:500, Chemicon) and α-actin (rabbit monoclonal antibody, 1:200, Sigma). After washing three times with PBS, cells were incubated with Alexa Fluor 488 (donkey anti-mouse) or Alexa Fluor 546 (goat anti-rabbit) conjugated secondary antibody (1:500; Chemicon) for 1h at room temperature in the dark. Finally, DNA was stained with Hoechst33342 (Beyotime, C1005) for 3 min. Negative controls were processed the same way, except that the primary antibodies were replaced with blocking buffer.

Pan S., Chen W., Liu X., Xiao J., Wang Y., Liu J., Du Y., Wang Y, & Zhang Y. (2015). Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT. PLoS ONE, 10(1), e0114423.

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Comprehensive Protein Analysis of Tissue Samples

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Tissue was homogenized using a Tissumizer from Tekmar in RIPA buffer and equal amounts of protein were separated by SDS-PAGE. The following antibodies were used for protein detection: MDM2 clone 2A10 (kind gift from Dr. Arthur Levine); MDM2 (AF1244) from R&D Systems; Gapdh (14C10), Pten (138G6), pAkt S473 (D9E) from Cell Signaling; Rela (C-20), Xiap (E-2), Slug (A-7) from Santa Cruz Biotechnology; p19Arf from Abcam; pMDM2 S395 from Thermo Fisher; and β-Actin (AC-15) from Sigma. Tissue specimens were stained with the following antibodies: B220 from BD Pharmingen; CD3, Myogenin (F5D), and Smooth Muscle Actin (1A4) from Dako; and Desmin from Abcam.

Comiskey DF J.r., Jacob A.G., Sanford B.L., Montes M., Goodwin A.K., Steiner H., Matsa E., Tapia-Santos A.S., Bebee T.W., Grieves J., La Perle K., Boyaka P, & Chandler D.S. (2017). A novel mouse model of rhabdomyosarcoma underscores the dichotomy of MDM2-ALT1 function in vivo. Oncogene, 37(1), 95-106.

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Western Blot Analysis of Mammalian Proteins

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Cell lysates prepared using mammalian protein extraction reagent (M-PER®; Pierce Biotechnology, Rockford, IL) were mixed with 2× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA), boiled for 5 min and separated on a precast 12% SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA). Proteins were transferred on PVDF membranes, blocked with 5% w/v skim milk and probed overnight (o/n) with antibodies against β-actin (Abcam, Cambridge, MA) and desmin (Abcam, Cambridge, MA) at 4°C with gentle shaking. Membranes were washed with tris-buffered-saline (TBS) containing 0.1% w/v tween and probed with goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX) and donkey anti-goat IgG-HRP (Abcam, Cambridge, MA) for 1 h at room temperature (RT) with gentle shaking before visualizing the protein bands using Pierce™ ECL Western Blotting substrate (Thermo Fisher Scientific, Waltham, MA).

Mahajan V., Gaymalov Z., Alakhova D., Gupta R., Zucker I.H, & Kabanov A.V. (2015). Horizontal Gene Transfer from Macrophages to Ischemic Muscles upon Delivery of Naked DNA with Pluronic Block Copolymers. Biomaterials, 75, 58-70.

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Immunostaining of Fibrin Scaffold Samples

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Fibrin scaffolds were fixed in 4% PFA for 2 h at RT, cryo-protected in 30% sucrose (4 °C, overnight), included in optimal cutting temperature (OCT) compound, and cut in slices of 10 µm thickness using a cryostat (CM 1950, Leica, Wetzlar, Germany). Slices were permeabilized with 0.1% Triton X-100 for 10 min and blocked with horse serum solution for 1 h. Samples were then stained for Desmin (1:100; Abcam) and MYH2 (1:50, Thermo Fisher Sci.), incubated overnight at 4°C, and subsequently incubated for 1 h at RT with Alexa Fluor ™ 488 goat anti-rabbit IgG (1:400; Thermo Fisher Sci.), VectaFluor™ anti-mouse IgG Dylight 594^® kit (Vector laboratories), and DAPI. Slices were also stained for FITC-conjugated CD90 and PE-conjugated CD105 (Beckman Coulter), incubated overnight at 4°C, and the cell nuclei were counterstained using DAPI. Separate images were acquired using identical settings of light intensity, exposure time, and gain using a fluorescence microscope (Eclipse Ti Nikon Corporation, Tokyo, Japan).

Scala P., Manzo P., Lamparelli E.P., Lovecchio J., Ciardulli M.C., Giudice V., Selleri C., Giordano E., Rehak L., Maffulli N, & Della Porta G. (2023). Peripheral blood mononuclear cells contribute to myogenesis in a 3D bioengineered system of bone marrow mesenchymal stem cells and myoblasts. Frontiers in Bioengineering and Biotechnology, 10, 1075715.

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Immunofluorescence Microscopy of Lung Cells

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WI-38 cells or lung microsections (5 μm) were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and blocked in 5% bovine serum albumin for 60 min. The cells or lung microsections were incubated with primary antibody at 4 ℃ overnight, and then incubated with secondary antibody for 1 h at room temperature after three washes with TBST. Nuclei were stained with DAPI for 3 min. Finally, the cells or lung microsections were observed under a laser confocal microscope (Leica TCS SP8; Leica, Wetzlar, Germany). Images from each sections were measured in a blinded manner and quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The following primary antibodies and dilutions were used for immunofluorescence microscopy experiments: α-SMA (1:50, ab7817; Abcam), desmin (1:50, ab15200; Abcam), Ki67 (1:100, 556003; BD Biosciences). The following secondary antibodies were used: anti-mouse-Alexa Fluor 647 (1:1000, Invitrogen), anti-rabbit-Alexa Fluor 488(1:1000, Invitrogen).

Zhang J.X., Lu J., Xie H., Wang D.P., Ni H.E., Zhu Y., Ren L.H., Meng X.X, & Wang R.L. (2019). circHIPK3 regulates lung fibroblast-to-myofibroblast transition by functioning as a competing endogenous RNA. Cell Death & Disease, 10(3), 182.

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Cardiac Necrosis Quantification in Mice

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Ventricular myocardium from T60A-β5 tg mice was fixed with 3.8% paraformaldehyde and processed for cryosectioning, as previously described [22 (link)]. To discern cardiomyocytes, the myocardial sections were stained for desmin (Abcam, Cambridge, MA) using indirect immunofluorescence. Necrotic cells were stained with a Fluro565-conjugated anti-mouse IgG antibody (Life Technologies, Grand Island, NY). Nuclear DNA was stained with DAPI. The fluorescence staining was visualized and imaged using a multi-laser fluorescence confocal microscope (Olympus Fluoview 500). ImagePro Plus software (Media Cybernetics, Rockville, MD) was used to quantify the percentage of the amount of red fluorescence (necrosis) to green fluorescence (cardiomyocytes) [22 (link)].

Ranek M.J., Zheng H., Huang W., Kumarapeli A.R., Li J., Liu J, & Wang X. (2015). Genetically induced moderate inhibition of 20S proteasomes in cardiomyocytes facilitates heart failure in mice during systolic overload. Journal of molecular and cellular cardiology, 85, 273-281.

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Cardiomyogenic Differentiation of MSCs

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Induction of MSC differentiation into cardiomyocytes was performed as previously described with slight modifications [27 (link)]. In brief, GFP-expressing MSCs were co-cultured with isolated neonatal rat cardiomyocytes at a ratio of 5000:5000 cells on a slide for 1 week, followed by immunofluorescence analysis. The medium was replenished every 3 days.
Differentiated MSCs were characterized by immunocytochemistry analysis as previously described [32 (link)]. In brief, cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked in 10% donkey serum (Beijing Solarbio Science & Technology Co., Ltd.). Cells then were incubated with antibodies against cTnT (1:400, Abcam) and desmin (1:100, Abcam) at 4 °C overnight. The cells were then further incubated with the appropriate secondary antibodies (Abcam) diluted at 1:100 for 1 h at room temperature, followed by another incubation with DAPI (Beijing Solarbio Science & Technology Co., Ltd.). Untreated rat MSCs and adult cardiomyocytes were used as negative and positive controls, respectively. The results of immunostaining were observed under fluorescence microscopy (Olympus FV1000).

Tong C., Li C., Xie B., Li M., Li X., Qi Z, & Xia J. (2019). Generation of bioartificial hearts using decellularized scaffolds and mixed cells. BioMedical Engineering OnLine, 18, 71.

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Isolation and Characterization of Smooth Muscle Cells

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The SMCs were isolated from SD rats, as described previously [47 (link),48 (link)]. To obtain PA and thoracic arterial SMCs (PASMCs and TASMCs), the artery was dissected from the lung and cardiac tissues. The arterial tissues were then cut into small pieces. After approximately 72 h, SMCs started to migrate out from the tissues. Cells were resuspended in DMEM (GIBCO, Paisley, UK) supplemented with 100 U/mL penicillin and 100μ g/mL streptomycin (PAN), 0.5 mM l-glutamine (GIBCO, Paisley, UK), and 10% fetal calf serum and incubated at 37 °C in 5% CO₂-95% air. The medium was changed then and thereafter every 2–3 days. To characterize SMCs, we used the smooth muscle cell–specific gene markers α-smooth muscle actin (Sigma-Aldrich, Saint Louis, MO, USA) and desmin (Abcam, Cambridge, MA,. USA). desmin was down-regulated at passage 3. Therefore, PASMCs were used before passage 2 for all of the in vitro experiments. The SMCs were studied from passages one to two. SMCs were characterized by immunocytochemical staining for α-smooth muscle actin and desmin.

Chang C.J., Huang C.C., Chen P.R, & Lai Y.J. (2020). Remodeling Matrix Synthesis in a Rat Model of Aortocaval Fistula and the Cyclic Stretch: Impaction in Pulmonary Arterial Hypertension-Congenital Heart Disease. International Journal of Molecular Sciences, 21(13), 4676.

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