A5114

Manufactured by Agilent Technologies

The A5114 is a high-performance liquid chromatography (HPLC) system offered by Agilent Technologies. It is designed for the separation, identification, and quantification of various chemical compounds in complex mixtures. The A5114 system provides precise and reliable performance for routine and advanced analytical applications.

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Lab products found in correlation

M0851, by Agilent Technologies (3 mentions) Ab6586, by Abcam (2 mentions) Mouse on mouse mom kit, by Vector Laboratories (1 mentions) Alexa fluor 647 secondary antibody, by Thermo Fisher Scientific (1 mentions) Zen software, by Zeiss (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Dia 310, by Dianova (1 mentions) K5007, by Agilent Technologies (1 mentions) M7020, by Agilent Technologies (1 mentions) Ab34710, by Abcam (1 mentions) Ab98952, by Abcam (1 mentions) S2369, by Agilent Technologies (1 mentions) Streptavidin hrp, by Biocare Medical (1 mentions) Mc120 hd microscope camera, by Leica (1 mentions) Las v 4, by Leica (1 mentions) Dm 1000 led microscope, by Leica (1 mentions) Ab52625, by Abcam (1 mentions) F3165, by Merck Group (1 mentions) Genetools software, by Syngene (1 mentions) 0.45 μm nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)

4 protocols using a5114

Lineage Tracing and Immunohistochemistry of Tumor Tissue

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Tumor tissues from the mice with R26^Dual lineage tracing were fixed in 4% paraformaldehyde overnight at 4 °C and equilibrated in 30% sucrose overnight at 4 °C. Tissues were then embedded in O.C.T. compound (TissueTek) and processed for 5-μm-thick cryosections. Sections were blocked for 1 h with 4% cold water fish gelatin (Aurion) and immunostained overnight at 4 °C with αSMA antibody (M0851, Dako, 1:100) or Fsp1 antibody (A5114, Dako, 1:100), followed by AlexaFluor647 secondary antibodies (Invitrogen). Staining for αSMA was performed with Mouse-on-Mouse (M.O.M.) kit (Vector Laboratories) following the manufacturer’s instructions. Slides were then mounted with DAPI-containing Vectashield Mounting Medium (Vector Laboratories), visualized under the LSM800 confocal laser scanning microscope, and analyzed with ZEN software (Zeiss). Immunohistochemical staining of formalin-fixed, paraffin-embedded sections were conducted using the following primary antibodies: CD31 (DIA310, Dianova, 1:100), Collagen IV (AB6586, Abcam, 1:100), and NG2 (AB5320, Chemicon/Millipore, 1:150), followed by fluorescence-labeled secondary antibodies (Jackson ImmunoResearch).

Chen Y., Kim J., Yang S., Wang H., Wu C.J., Sugimoto H., LeBleu V.S, & Kalluri R. (2021). Type I collagen deletion in αSMA+ myofibroblasts enhances immune suppression and accelerates progression of pancreatic cancer. Cancer cell, 39(4), 548-565.e6.

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Immunostaining of Lung Tissue Sections

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Lung tissue sections were deparaffinised in xylene and antigen retrieval carried out using target retrieval citrate buffer pH 6.0 (Dako S2369) for 15 min. Tissues were immunostained with polyclonal rabbit anti-human S100A4 (1:1000; Dako A5114), mouse monoclonal VE-cadherin (CD144) (1:150; Thermofisher 14144982), vimentin, monoclonal mouse (1:200; Dako M7020), mouse monoclonal anti-N-cadherin (1:100; Abcam ab98952), monoclonal mouse anti-α-SMA (1:500, M0851, Dako), collagen type I (1:200, Abcam ab34710), rabbit polyclonal and collagen type IV (1:200, Abcam ab6586), rabbit polyclonal for 60 min followed by secondary HRP rabbit/mouse antibodies (Dako K5007) treatment for a further 30 min. The protein markers were visualised as brown after adding DAB substrate and counterstained for the nucleus with haematoxylin.

Gaikwad A.V., Lu W., Dey S., Bhattarai P., Chia C., Larby J., Haug G., Myers S., Jaffar J., Westall G., Singhera G.K., Hackett T.L., Markos J., Eapen M.S, & Sohal S.S. (2022). Vascular remodelling in idiopathic pulmonary fibrosis patients and its detrimental effect on lung physiology: potential role of endothelial-to-mesenchymal transition. ERJ Open Research, 8(1), 00571-2021.

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Histopathological Analysis of Metastases

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For paraffin‐fixed samples, mouse tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. In search for metastases, 20 sections for each tissue type (lung or liver) per mouse were randomly selected from the serial sectioning (with 100 μm interval) of the entire lung or liver lobe. Sections were processed for hematoxylin and eosin (H&E) staining. Microscopic metastases were examined in H&E‐stained tissue sections of the liver and lung. Images were captured with a Leica DM 1000 LED microscope and an MC120 HD Microscope Camera with Las V4.4 Software (Leica). Formalin‐fixed, paraffin‐embedded sections were processed for immunohistochemical staining as previously documented (Zheng et al, 2015). Sections were incubated with primary antibodies: αSMA (M0851, Dako, 1:100), CK19 (ab52625, Abcam, 1:200), or Fsp1 antibody (A5114, Dako, 1:100), then biotinylated secondary antibodies, and streptavidin HRP (Biocare Medical). For all immunolabeling experiments, sections were developed by DAB and counterstained with hematoxylin.

Chen Y., LeBleu V.S., Carstens J.L., Sugimoto H., Zheng X., Malasi S., Saur D, & Kalluri R. (2018). Dual reporter genetic mouse models of pancreatic cancer identify an epithelial‐to‐mesenchymal transition‐independent metastasis program. EMBO Molecular Medicine, 10(10), e9085.

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Immunoblotting of Mybpc3-KI Mouse Heart Proteins

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Proteins were extracted from EHTs and powdered whole heart lysate of Mybpc3-KI mice and immunoblotting was performed as described before (Friedrich et al., 2012 (link)). Proteins were loaded on 7–10% acrylamide/bisacrylamide (37.5:1 or 29:1 BioRad) and 16.5% tris-tricine gels and then blotted onto a 0.45-μm nitrocellulose membrane (Life Technologies). Membranes were blocked in 5% milk and incubated with antibodies against S100A4 (anti-S100A4 rabbit-polyclonal, Dako A5114, 1:500), ß-actin (anti-ß-actin mouse-monoclonal, 1:20,000), GAPDH (anti-GAPDH mouse-monoclonal, HyTest 5G4-6C5, 1:5,000), FLAG (anti-FLAG mouse-monoclonal, Sigma F3165, 1:5,000), V5 (anti-V5 mouse-polyclonal, Invitrogen PA1-993, 1:1,000), GFP (anti-GFP rabbit-polyclonal, Abcam ab6556, 1:2,000), and total ERK (anti-Total ERK rabbit-polyclonal, Cell Signaling #9102, 1:2,000). Secondary antibodies were coupled to HRP (Sigma). Signals were detected by ECL Plus Western blotting detection system substrate (Amersham GE Healthcare Life Sciences, Munich, Germany). Quantification of the signal was determined using Gene Tools software (Syngene, Cambridge, UK). S100A4-protein levels were normalized to ß-actin, GAPDH, or total ERK, as mentioned above.

Braumann S., Thottakara T., Stücker S., Reischmann-Düsener S., Krämer E., Groß J., Hirt M.N., Doroudgar S., Carrier L, & Friedrich F.W. (2018). S100A4 as a Target of the E3-Ligase Asb2β and Its Effect on Engineered Heart Tissue. Frontiers in Physiology, 9, 1292.

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