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9 protocols using anti desmin

1

Profiling Heat-Induced Molecular Changes

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Changes in the molecular properties of the heat-exposed cells were investigated using ICC, western blotting, and qRT-PCR. Five different primary antibodies (DAKO, Denmark), anti-E-cadherin (1 : 300), anti-CK8/18 (1 : 200), anti-vimentin (1 : 1000), anti-desmin (1 : 150), and anti-p53 (1 : 300), were employed for immunocytochemistry (ICC). The antibodies were applied to quantify the changes in EMT-related properties or demonstrate the cell death mechanism.
The changes in protein expression were further determined by western blotting. The expression of heat shock protein 70 (Hsp70, 1 : 1000, Merck Millipore, Germany), Hsp90 (1 : 500, Santa Cruz, CA, USA), E-cadherin (41 : 1000, Merck Millipore), and vimentin (1 : 1000, Merck Millipore) were quantified by comparing to that of β-actin (1 : 1000, Merck Millipore).
EMT-related genes were quantified using qRT-PCR. Primer sequences, including HSPA1A (Hs00359163_s1), CDH1 (Hs01023894_m1), VIM (Hs00958111_m1), TWIST1 (Hs01675818_s1), and GADPH (Hs02758991_g1), were purchased from Thermo Fisher Scientific. See ESI for additional details.
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2

Antibody Validation for Protein Analysis

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Antibodies used for this study include anti-APE1 (Santa Cruz, sc-55498), anti-DCN (Santa Cruz, sc-22753), anti-PGC1-alpha (Santa Cruz, sc-13067), anti-SOD3 (Santa Cruz, sc-67088), anti-NRF2 (ABcam, AB31163), anti-Ki67 (Dako IR626), and anti-desmin (Dako IR606). Antibodies were either validated by western blot analysis or by The Markey Cancer Center Biospecimen and Tissue Procurement Shared Resource Facility (BSTP SRF).
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3

Histological Analysis of Embryonic Lung Development

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Embryos and tissues were fixed in 10% formalin overnight, dehydrated in 100% ethanol, and embedded in paraffin. 8-µm-thick sections were used for hematoxylin-eosin, periodic acid-Schiff (PAS), trichrome, and immunohistochemistry staining. The following primary antibodies were used for immunostaining: anti-LYVE1 (R&D Systems), anti-PROX1 (Abcam), anti-PECAM1 (R&D Systems; clone: 693102), anti–pro-surfactant protein C (pro-SPC; EMD Millipore), anti-Clara cell 10 (CC10; Santa Cruz Biotechnology, Inc.), anti-Podoplanin (Abcam), anti–Caveolin-1 (BD), anti-PDGFRα (Cell Signaling Technology), anti-PDGFRβ (Cell Signaling Technology), anti-SM22α (Abcam), anti-WT1 (Santa Cruz Biotechnology, Inc.), anti-Vimentin (Santa Cruz Biotechnology, Inc.), anti-Desmin (Dako), and anti-NG2 (EMD Millipore). Detailed histology procedures can be found on the University of Pennsylvania Molecular Cardiology Research Center Histology and Gene Expression Core website. Microscopic images were taken on a microscope (Eclipse 80i; Nikon) connected to a camera (DS-Ri1; Nikon). Alveolar wall thickness (averaging 50–100 measurements per embryo) and alveolar area (averaging the total alveolar area in 5–15 vision fields per embryo) were performed in Elements software (Nikon) using a 40× objective.
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4

Immunohistochemical Analysis of Tissue Samples

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The following antibodies were utilized for immunohistochemistry on formalin fixed paraffin embedded tissue. Anti-pancytokeratin (mouse monoclonal AE1/AE3; catalog # NB600-1322) was obtained from Novus Biologicals (Littleton, CO, USA). Anti-S100 protein (rabbit polyclonal; catalog # Z0311) was obtained from Dako (Carpinteria, CA, USA). Anti-SMA (mouse monoclonal 1A4; catalog # A2547) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-desmin (mouse monoclonal D33; catalog # M0760) was obtained from Dako. Anti-myogenin (mouse monoclonal F5D; catalog# M3559) was obtained from Dako. Anti phospho-AKT Ser473 (rabbit monoclonal D9E; Cell Signaling Technology catalog #4060 (Danvers, MA, USA). Anti-PTEN (rabbit monoclonal D4.3 XP; catalog# 9188) was obtained from Cell Signaling Technology. Anti-CD45 (rabbit polyclonal; catalog# 10558) was obtained from Abcam (Cambridge, MA, USA). Anti-PD-L1 (rabbit polyclonal; catalog# 17952-1-AP) was obtained from Proteintech (Rosemont, IL, USA). Dilutions utilized for immunohistochemistry are as follows pan-cytokeratin (1:1600), S100 protein (1:4000), SMA (1:2000), desmin (1:50), myogenin (1:50), phospho-AKT (1:50), PTEN (1:50), CD45 (1:100), and PD-L1 (1:50). Horseradish peroxidase-conjugated secondary antibodies were obtained from Dako.
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5

Renal Tissue Immunohistochemistry Assessment

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Immunohistochemistry studies were performed on fragments of 4-µm-thick paraffinized renal tissue mounted on glass slides coated with Entellan™ (Merck Millipore). Kidney sections were incubated overnight at 4 °C with the following primary antibodies: anti-desmin (1:100, Dako, Denmark), anti-vimentin (1:200, Dako, Denmark) and anti-alpha-smooth muscle actin (α-SMA, 1:200, Dako, Denmark). Secondary antibodies were used according to the primary antibody. The sections of renal tissue were revealed with avidin-biotin-peroxidase complex (Vector Laboratories, USA) and 3,3-diaminobenzidine (DAB) (Sigma, USA). The sections were counterstained with methyl green. The evaluations of the immunoreaction for desmin (50 glomeruli), vimentin (30 cortical fields), and alpha-smooth muscle actin (α-SMA, 30 cortical and 20 outer medulla interstitial fields) were performed by computational morphometry using ImageJ 1.44p program (National Institute of Health, USA) and the results expressed as the percentage of the marked area for each of these antibodies. The results corresponding to the desmin immunostaining area were corrected for the corresponding glomerular tuft area and vimentin and α-SMA for the total area of the cortex and medulla per field. The average values per sample were calculated and expressed as a percentage [27 ].
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6

Immunohistochemical Tissue Analysis

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Tissue samples were fixed in 4% formalin and were paraffin-embedded. Four
micrometer routinely processed paraffin sections were performed for standard
coloration by hemalun and eosin and for immunohistochemistry using a VentanaBenchMark
XT immunostainer (Ventana medical system, Tucson, Arizona). Immunostaining was
performed with the following primary antibodies: anti-cytokeratin (Clone AE1/AE3,
Ménarini, 1/400), anti-actin (Clone 1A4, Dako, 1/300), anti-desmin (Clone D33,
Dako, 1/100) and anti-PS100 (polyclonal rabbit antibody, Leica, 1/400).
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7

Quantitative Dysferlin Analysis in Muscle Tissue and Cells

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Transversal muscle tissue cryosections of 6 μm were fixed for 5 min in acetone at −20°C and blocked with 1% BSA in PBS for 45 min at room temperature. Sections were incubated overnight at 4°C with the primary ROMEO anti-Dysferlin rabbit monoclonal antibody (Abcam, Cambridge, UK, 1:100). Sections were incubated with the secondary goat anti-rabbit Cy3-conjugated antibody (Dianova, Hamburg, Germany, 1:500) for 45 min at room temperature. Nuclei were stained with Hoechst. The sections were mounted with AquaMount (Polysciences, Hirschberg, Germany). Cells were seeded on glass-bottom plates (ibidi, Martinsried, Germany) and fixed with 3.7% formaldehyde for 10 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. Cells were blocked with 1% BSA in PBS for 1 hr at room temperature and incubated overnight at 4°C with two primary antibodies: mouse monoclonal antibody anti-Desmin (Dako, Jena, Germany; 1:100) and rabbit monoclonal antibody ROMEO anti-Dysferlin (Abcam, 1:150). Cells were incubated for 1 hr at room temperature with the following secondary antibodies (both diluted to 1:500): polyclonal Alexa 568 donkey anti-rabbit (Life Technologies) and polyclonal Alexa 647 goat anti-mouse (Life Technologies). Nuclei were stained with Hoechst.
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8

Immunohistochemical Staining of Muscle Proteins

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Immunohistochemical staining on 4% paraformaldehyde fixed sections was carried out using the following antibodies: anti-desmin (1:100 M0760, clone D33 mouse monoclonal DAKO), anti-TDP43 (1:200 10784-2-AP rabbit monoclonal Proteintech), anti-myotilin (1:100 mouse monoclonal Novacastra Leica), anti-p62 (1:100 gp62-c guinea-pig polyclonal; Progen), anti-VCP (1:100 MA3-004 mouse; ThermoScientific). Specific secondary Alexa 488/546/555 antibodies (1: 1500; Invitrogen Life Technology) were used and visualized under a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).
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9

Histological Analysis of Liver Fibrosis

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Liver samples were formalin fixed, paraffin embedded and 4 μm sections obtained. Hematoxylin and eosin (H&E) staining was performed for structural analysis. Sirius Red was used to deter-mine collagen deposition; sections were stained with Sirius Red solution (saturated picric acid containing 0.1% Direct Red 80) to visualize collagen. The red stained area per total area was deter-mined using AxioVision software and values are expressed as the mean of 10 fields at 20× magnification obtained with a Zeiss Axiovert 135 microscope.
Immunohistochemical staining of aSMA, Von Willebrand factor (VWF) and desmin were performed with anti-aSMA (½00, Abcam), anti-VWF (1/100, Dako) and anti-desmin (1/100, Dako). Bound antibodies were visualized with Dako REAL™ EnVision™ Detection System Peroxidase/DAB + kit, and slides were counterstained with hematoxylin. Fifteen images at 20 x magnifications were captured for immunohistochemical quantification with a Zeiss Axiovert 135 microscope and quantified with ImageJ software (NIH).
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