Four μm tissue sections were deparaffinized and blocked for endogeneous peroxidase activity by immersion in 0.3% H2O2 in methanol for 20 minutes. Antigen retrieval was performed in Tris/EDTA buffer (10 mM/1mM; pH 9.0) for 20 minutes at 100°C. After cooling for 10 minutes and washing in PBS pH 7.4, nonspecific binding sites were blocked with Serum Free Protein Block (Dako, Glostrup, Denmark) for 10 minutes followed by 1 hour incubation with SMAD4 antibody (Santa Cruz technology, Santa Cruz, CA, USA) at room temperature. Antibody binding was visualized using the BrightVision poly-HRP detection system (Immunologic, Duiven, the Netherlands) with 3,3-diamino-benzidine as chromogen. Sections were counterstained using hematoxylin, dehydrated and coverslipped using Pertex. Pancreas cancer tissue sections were used as negative control, showing no SMAD4 staining [28 (link)]. Control slides in which the primary antibody was omitted, were included. All sections were evaluated using a binocular microscope (Olympus BX 51, Zoeterwoude, the Netherlands).
Smad4 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The SMAD4 antibody is a laboratory reagent used in research applications. It is a protein-specific antibody that recognizes and binds to the SMAD4 protein, which is a key mediator in the transforming growth factor-beta (TGF-β) signaling pathway. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression, localization, and function of SMAD4 in biological systems.
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2 protocols using smad4 antibody
1
Immunohistochemical Detection of SMAD4
2
SMAD4 Expression in Activated T Cells
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Naive T cells were activated for 24 hours and then resuspended in culture medium at 1 million/ml. One hundred microliters of each cell suspension was added to a slide chamber and spun down onto the slide using a cytocentrifuge (800 rpm/3 min). The cells were fixed on slide with 4% paraformaldehyde, permeabilized with 0.01% Trion X-100, and blocked with goat serum and stained with SMAD4 antibody (Santa Cruz Biotechnology, catalog no. sc-7966) overnight. The slides were washed and then incubated with Alexa Fluor 488– or Alexa Fluor 594–conjugated goat anti-mouse immunoglobulin G (IgG) (BioLegend) secondary antibody for 1 hour at room temperature and lastly mounted with mounting medium containing 4′,6-diamidino-2-phenylindole. The images were obtained by LSM780 fluorescence microscope (Zeiss).
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