Applications of Immunocytochemistry

Recombinant Anti-Smad3 (phospho S423 + S425) antibody [EP823Y] - BSA and Azide free (ab172202)
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Visualize and quantify cell-cell interactions overtime in complex co-culture models, revealing insight into the interplay of cells. Measure the dynamic changes in cell surface proteins in response to inflammatory stimuli to provide additional insight into regulation of the immune-cell signaling pathways in tumors.

Figure 1. Automated analysis of IncuCyte FabFluor reagent fluorescence reveals cell type-, time- and concentration-dependent increase in PD-L1 expression right. Click image to enlarge.

Reveal the Dynamics of Surface Protein Expression with a New Approach to Immunocytochemistry

Rapid, single step labeling plus automated acquisition and analysis of images from up to six well plates in parallel to get answers faster. Easily link long-term changes in protein abundance and distribution to changes in morphology and function in real time. Figure 2. Multiplex cell surface marker, phagocytic activity, and proliferation measurements plus visualize morphology to study differentiation right. PMA showed a marked change in cell morphology HD-phase contrast images compared to media alone or vitamin D3 treated cells.

The kinetic graphs highlight differential and time-dependent surface protein expression in response to the various treatments. Interestingly, PMA, but not media or vitamin D3, yields a decrease in cell proliferation confluence and concordant increase in phagocytic potential as measured by efferocytosis of apoptotic Jurkat cells labeled with IncuCyte pHrodo Red cell labeling Kit.

Observe and quantify cell-cell interactions via cell surface protein expression markers, enabling insight into complex co-culture models. Figure 3. Quantification of the interaction overlay, yellow mask in images reveals a marked increase in the interaction of activated effector cells with the target cells indicating cell engagement for immune killing of tumor cells as shown in the bar graphs.

Confirm cell-cell interactions in an immune cell killing model over time. The IncuCyte mouse FabFluor reagents are non-perturbing and produce specific long term labeling for powerful analysis of surface protein dynamics on live cells over hours or days. Figure 4. Antibodies should undergo a stringent validation procedure to ensure that the antibody detects the target accurately.

You must also ensure the signal you are detecting is specific. An important consideration in any experiment is the inclusion of appropriate controls. Positive and negative controls instill confidence that your antibody is detecting its intended target. PLK1 G4 Rabbit mAb IHC analysis of paraffin-embedded human colon carcinoma using with step-by-step reagent substitution, as indicated, demonstrating that reagents can make the difference between poor staining and publishable results.

Suboptimal IHC staining is frequently resolved by adjusting relatively few variables. Adjustments to key steps within the protocol, such as antigen retrieval, can often resolve common issues, such as little or no signal and high background. The primary antibody is a critical component of any IHC assay and has a direct effect on data quality. The determination of target specificity in IHC analysis requires multiple validation steps. In-house validation allows our scientists to provide expert advice that will save you time and accelerate your results.

CST scientists use a variety of methods, as appropriate, to validate each IHC-recommended antibody, ensuring that the staining you observe with each antibody is specific. Validation steps include: Western blot analysis to demonstrate the appropriate molecular weight s , with minimal crossreacting bands.

Paraffin-embedded cell pellets of known target expression levels are used to verify target specificity. Antibody performance is assessed in relevant models of cancer, including but not limited to human cancer tissue arrays, mouse models, and xenografts from cell lines with known target expression levels.

Tissue sections and cell pellets are subjected to phosphatase treatment to verify target phospho-specificity. Blocking peptides verify specificity and rule out Fc-mediated binding and other nonspecific staining. Thorough lot testing ensures the reproducibility necessary for accurate IHC results. The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.

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Immunohistochemistry (IHC) Antibodies

Purification Purification Process designed to isolate a single type of protein from a complex mixture. Antiserum to surface antigens as a marker for cultured rat lung endothelial cells 1. Visualization of central noradrenergic neurons in thick sections by the unlabeled antibody method: a transmitter-specific Golgi image. Pronase pretreatment of tissue sections enhances sensitivity of the unlabelled antibody-enzyme PAP technique. Carcinoembryonic antigen in histopathology: immunoperoxidase staining of conventional tissue sections.

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This experiment was performed under reducing conditions using the kDa separation system. The cells Product Details Summary. Learn about the Innovator's Reward. The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Immunohistochemistry vs Immunocytochemistry

Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer pH 6. In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Simple Western. Western Blot. Chinese hamster data from customer review. Sordaria macrospora reactivity reported in scientific literature PMID: Storage Store at 4C short term. Aliquot and store at C long term. Avoid freeze-thaw cycles. Actins are highly conserved proteins that are involved in various types of cell motility, and maintenance of the cytoskeleton.

In vertebrates, three main groups of actin isoforms, alpha, beta and gamma have been identified.

The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins co-exist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility. This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt. We have publications tested in 1 application: WB.

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  1. Understand Protein Expression in Tissue with Immunohistochemistry (IHC).
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Ageing Dev. PPFIA1 drives active alpha5beta1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis. Nat Commun.