Desmin clone d33

Manufactured by Agilent Technologies

Sourced in United States, Germany

Desmin (clone D33) is a laboratory reagent used for the identification and visualization of desmin, a cytoskeletal protein found in muscle cells. This reagent is intended for research use only and not for use in diagnostic procedures.

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9 protocols using desmin clone d33

Immunohistochemical Profiling of FFPE Tissue

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Formalin-fixed paraffin-embedded tissue (FFPE) sections were immunostained with rabbit primary antibodies against NF-κB p65, alpha smooth muscle actin (α-SMA) and desmin. In the immunohistochemical (IHC) protocol, 4 μm thick FFPE slides were deparaffinized and rehydrated with xylene and ethanol. Antigen retrieval was applied by boiling the slides in the triplicates (Cell Marque, Rocklin, CA, USA) in a microwave at 95 °C for 10 min. The slides were then incubated with 10% protein-blocking normal goat and rabbit sera (Dako, Glostrup, Denmark) for 30 min to reduce non-specific binding. Antigen expression was detected by primary antibodies against rabbit polyclonal NF-κB p65 (clone ab86299, 1 in 200 dilution; Abcam, Cambridge, MA, USA), α-SMA (clone 1A4, 1 in 200 dilution; Dako) and desmin (clone D33, 1 in 200 dilution; Dako) for 1 h. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (KYB, New Taipei City, Taiwan) for 15 min. Afterward, primary antibodies were detected using a REAL Envision kit (Dako) using diaminobenzidine as a chromogenic substrate. The slides were finally counterstained with hematoxylin (Muto Pure Chemicals, Tokyo, Japan) for 15 s. Substitution of primary antibody with antibody diluent was performed as a negative control.

Hsueh C.S., Wu C.H., Shih C.H., Yeh J.L., Jeng C.R., Pang V.F., Chiou H.Y, & Chang H.W. (2019). Role of nuclear factor-kappa B in feline injection site sarcoma. BMC Veterinary Research, 15, 365.

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Immunohistochemical Analysis of FFPE Tumors

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Four µm-thick serial sections were cut from the formalin-fixed paraffin-embedded (FFPE) tumour blocks and were used for haematoxylin and eosin (HE) staining and immunohistochemical analysis. HE staining was performed according to standard protocols.
Slides of tumour were de-paraffinized in xylene, hydrated in alcohol and baked in a microwave. Endogenous peroxidase was blocked. Immunohistochemical analysis was performed in all cases with the following antibodies: desmin (clone D33, Dako), myogenin (clone LO26, Novacastra), transgelin (clone D33, LSBio, Seattle, WA, United States) and caldesmone (clone h-CD, Dako). Appropriate positive and negative controls for each antibody were included. Pictures were captured using a Zeiss Cell Observer Microscope (Zeiss).

Delespaul L., Gélabert C., Lesluyes T., Le Guellec S., Pérot G., Leroy L., Baud J., Merle C., Lartigue L, & Chibon F. (2020). Cell–cell fusion of mesenchymal cells with distinct differentiations triggers genomic and transcriptomic remodelling toward tumour aggressiveness. Scientific Reports, 10, 21634.

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Comprehensive Immunohistochemistry Panel for Tumor Diagnosis

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Cases were identified in the consultation files of the authors. The tissue specimens were fixed in formalin and processed routinely for histopathology. Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, Arizona, USA) and the following antibodies: vimentin (V9, 1:100, Dako), keratin cocktail (clone AE1/AE3, 1:40, Zytomed, Berlin, Germany), p63 (SSI6, 1: 100, DCS), desmin (clone D33, 1:250, Dako), alpha smooth muscle actin (clone 1A4, 1:200, Dako), HMB45 (clone HMB45, 1:50, Enzo), Melan A (clone A103, 1:50, Dako), CD34 (clone BI-3C5, 1:200, Zytomed), ERG (EPR3864, prediluted, Ventana), CD31 (clone JC70A, 1:20, Dako), S100 protein (polyclonal, 1:2500, Dako), SOX10 (polyclonal, 1:25, DCS), PAX8 (polyclonal rabbit anti-PAX8, 1:50, Cell Marque), NSE (clone BBS/NC/VI-H1, 1:300, Dako), TTF1 (clone 8G7G3/1, 1:500, Zytomed Systems, Berlin, Germany), Napsin A (MRQ-60, ready-to-use, Medac), calretinin (polyclonal, 1:100, Zytomed), alpha-inhibin (clone R1, 1:50, Serotec), GFAP (Clone GFA, 1/1000, DakoPatts, Denmark), EMA (clone E29, 1:200, Dako), STAT6 (clone S-20, 1:1000, Santa Cruz Biotechnology), TFE3 (clone MRQ-37, 1:100, Cell Marque), Cathepsin-K (clone 3F9, 1:50, Abcam) and Ki67 (clone MiB1, 1:100, Dako).

Agaimy A., Stoehr R., Michal M., Christopoulos P., Winter H., Zhang L., Stenzinger A., Michal M., Mechtersheimer G, & Antonescu C.R. (2021). Recurrent YAP1-TFE3 Gene Fusions in Clear Cell Stromal Tumor of The Lung. The American journal of surgical pathology, 45(11), 1541-1549.

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Comprehensive Immunohistochemistry Panel for Sinonasal Tumors

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Immunohistochemistry for CD99 (clone 12E7; Leica, Buffalo Grove, IL; prediluted), pancytokeratin (PCK26; Ventana, Tuscon, AZ; prediluted); p40 (Ab-1; Oncogene Research Products, Cambridge, MA; 1:2000 dilution), synaptophysin (clone 27G12; Leica Microsystems; prediluted); chromogranin (clone LK2H10; Ventana; prediluted); S100 protein (clone 4C4.9; Ventana; prediluted), muscle specific actin (clone HHF35; Ventana, prediluted); desmin (clone D33, Dako, Carpinteria, CA; 1:100 dilution), NUT-1 (clone C52B1, Cell Signaling Technologies, Inc., Danvers, MA, 1:50 dilution), and p16 (clone INK4a; MTM Laboratories, Heidelberg, Germany); was performed on five-micron sections utilizing standard protocols on a Ventana Benchmark XT autostainer. In addition, in situ hybridization for high-risk types of human papillomavirus was also performed utilizing the Ventana HR HPV III probe set that captures HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 66. The sinonasal tissue microarray was also stained with CD99.

Bishop J.A., Alaggio R., Zhang L., Seethala R.R, & Antonescu C.R. (2015). Adamantinoma-like Ewing Family Tumors of the Head and Neck: A Pitfall in the Differential Diagnosis of Basaloid and Myoepithelial Carcinomas. The American journal of surgical pathology, 39(9), 1267-1274.

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Immunohistochemical Analysis of Tumor Markers

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Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue using the Dako Envision Plus detection system (Dako, Carpinteria, CA, USA) with controls. The antibodies used included MDM2 (clone SMP14, ready-to-use; Abcam, Cambridge, UK), CDK4 (clone EP180, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), S-100 protein (clone 4C49, 1:100; Abcam, Cambridge, UK), CD34 (clone QBEnd 10, 1:100; Abcam, Cambridge, UK), desmin (clone D33, 1:100; Dako, Carpinteria, CA, USA), smooth muscle actin (SMA) (clone 1A4, 1:100; Dako, Carpinteria, CA, USA), H-caldesmon (clone h-CD, 1:100; Dako, Carpinteria, CA, USA), and p53 (clone Do-7, ready-to-use; Dako, Carpinteria, CA, USA).

Xie Y., Jing W., Zhao W., Peng R., Chen M., Lan T., Peng H., He X., Chen H., Zhang Z, & Zhang H. (2022). Primary intrathoracic liposarcomas: A clinicopathologic and molecular study of 43 cases in one of the largest medical centers of China. Frontiers in Oncology, 12, 949962.

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Isolation of Tumor and Normal Gland Cells

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Crypts were isolated from tumor and normal tissues, as described previously, to obtain pure tumor glands separately from the surrounding stromal tissues (23 (link)–25 (link)). Tumor glands were isolated from the solid tumor region involved in the invasion front, and this involvement was confirmed using tissue sections prepared for the pathological diagnosis. Gland cells were obtained from the tumor tissues after careful separation of the stromal cells (i.e. CAFs) adjacent to the glands, performed under a dissecting microscope. Cells from normal fallopian tube tissue and normal fibroblasts within the Fallopian tubes were also obtained as controls. Paraffin-embedded tissue sections of the isolated samples were routinely processed to confirm the histology. Immunostaining using antibodies against smooth muscle actin (clone 1A4; Dako) and desmin (clone D33; Dako) was performed in the stromal cells to confirm the exclusive presence of fibroblasts, determined according to negative staining of smooth muscle actin and positive staining of desmin. However, we could not exclude the possibility of stromal cell contamination with other non-epithelial cells, such as inflammatory and vessel cells. Representative images are shown in Fig. 1.

Sato C., Osakabe M., Nagasawa T., Suzuki H., Itamochi H., Baba T, & Sugai T. (2020). Genome-wide analysis of microRNA to evaluate prognostic markers in isolated cancer glands and surrounding stroma in high-grade serous ovarian carcinoma. Oncology Letters, 20(6), 338.

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Histopathological and Immunohistochemical Analysis

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The surgical specimen was fixed in formalin and embedded routinely for histopathological evaluation. Hematoxylin and eosin (H&E) stained slides were prepared for histopathological examination and immunohistochemical staining was performed on 1 µm thick sections using an automated platform (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, Arizona, U.S.A.) and the following antibodies: CD34 (clone QBEND-10; 1:50, Immunotech), smooth muscle actin (clone 1A4, 1:400, Dako), desmin (clone D33, 1:50, Dako), MyoD1 (clone 5.8A, 1:50, Dako), myogenin (clone F5D, 1:50, Dako), S100 (clone 4C4.9, 1:3000, Zytomed), MDM2 (clone IF2, 1:50, Calbiochem), CDK4 (clone DCS-156, 1:100, Zytomed), Retinoblastoma-1 (clone G3-245, 1:100, BD Pharmingen), CD10 (clone 56C6, 1:20, Zytomed), STAT6 (clone S-20, 1:1000, Santa Cruz) and SATB2 (clone EPNCIR130A, 1:200, abcam).

Erber R., Preidl R., Stoehr R., Haller F., Hartmann A., Kesting M, & Agaimy A. (2021). DICER1-Mutated Botryoid Fibroepithelial Polyp of the Parotid Duct: Report of the First Case. Head and Neck Pathology, 16(2), 573-580.

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Comprehensive Immunohistochemical Analysis of Tissue Samples

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The biopsy specimen was fixed in buffered formalin and embedded in paraffin for routine histological examination. Immunohistochemical studies were performed on 3-µm sections cut from paraffin blocks using a fully automated system ("Benchmark XT System", Ventana Medical Systems Inc., 1910 Innovation Park Drive, Tucson, Arizona, USA) and the following antibodies: pan cytokeratin (clone AE1/AE3, 1 : 40, Zytomed, Berlin, Germany), vimentin (V9, 1 : 100, Dako, Hamburg, Germany), CK7 (clone OV-TL, 1 : 1000, Biogenex), CK18 (clone CY-90, 1 : 500, Sigma), CK5 (clone XM26, 1 : 50, Zytomed), TTF-1 (clone 8G7G3/1, dilution, 1 : 500, Zytomed), ERG (clone EPR3864, prediluted/ ready to use, Ventana Medical Systems), CD31 (clone JC70A, 1 : 20, Dako), CD10 (clone 56C6, 1 : 20, Dako), p63 (clone SFI-6, 1 : 100, DCS), desmin (clone D33, 1 : 250, Dako), α-smooth muscle actin (clone 1A4, 1 : 200, Dako), S100 protein (polyclonal, 1 : 2500, Dako), CD34 (clone BI-3C5, 1 : 200, Zytomed), CD30 (clone Ber-H2, 1 : 40, Zytomed), MUC4 (clone EP256, 1:500, Epitomics), TLE1 (polyclonal, 1 : 200, Santa Cruz), STAT6 (clone sc-621, 1 : 1000, Santa Cruz Biotechnology), and SMARCB1 (INI1) (MRQ-27, 1 : 50, Zytomed), according to the manufacturer instructions.

Agaimy A., Duell T, & Morresi-Hauf A.T. (2017). EWSR1-fusion-negative, SMARCB1-deficient primary pulmonary myxoid sarcoma. Polish journal of pathology : official journal of the Polish Society of Pathologists, 68(3).

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Immunohistochemical Profiling of Tumor Specimens

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All tumour specimens had been fixed in buffered formalin overnight and processed routinely for histological assessment. Immunohistochemistry, on 3 μm sections cut from paraffin blocks, was performed using a fully automated Benchmark XT System (Ventana Medical Systems, Tucson, AR, USA) and the following antibodies: pan-cytokeratin (clone AE1/AE3, 1:40, Zytomed); CK7 (OV-TL, 1:1000, Biogenex); vimentin (V9, 1:100, Dako); desmin (clone D33, 1:250, Dako); α-smooth muscle actin (clone 1A4, 1:200, Dako); h-caldesmon (clone h-CD, 1:100, Dako); protein S-100 (polyclonal, 1:2500, Dako); CD34 (clone BI-3C5, 1:200, Zytomed); ERG (EPR3864, prediluted, Ventana); myogenin (clone F5D, 1:50, Dako); and STAT6 (clone sc-621, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Haller F., Knopf J., Ackermann A., Bieg M., Kleinheinz K., Schlesner M., Moskalev E.A., Will R., Satir A.A., Abdelmagid I.E., Giedl J., Carbon R., Rompel O., Hartmann A., Wiemann S., Metzler M, & Agaimy A. (2016). Paediatric and adult soft tissue sarcomas with NTRK1 gene fusions: a subset of spindle cell sarcomas unified by a prominent myopericytic/haemangiopericytic pattern. The Journal of pathology, 238(5).

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