Answering your questions: How to detect glycan expression in tissue

Answering your questions: How to detect glycan expression in tissue
Posted in: Tips and Tricks

Glycosylation—the addition of carbohydrate chains to proteins and lipids—plays an essential role in cellular function and communication. Changes in glycosylation are relevant to physiology and pathophysiology in many research fields, including cancer biology, immunobiology, and neuroscience.  

Vector Laboratories has developed Glysite Scout Glycan Screening Kits to empower discoveries in the field of glycobiology. Glysite Scout kits offer a curated panel of lectins, optimized reagents, and protocols for detecting glycans in tissue sections using fluorescent probes. Double labeling involving immunofluorescence and fluorescent glycan detection is also possible. Versatile and easy-to-use glycan detection tools can empower biomarker discovery in normal and disease states, contributing to the identification of new therapeutic targets.   

The Scientist recently hosted a webinar featuring Dr. August Estabrook, a Senior Scientist at Vector Laboratories. Dr. Estabrook covered the basics of glycan screening and focused on how to use Glysite Scout to detect glycan expression in tissue sections. You can watch the entire on-demand webinar, or keep reading for tips & tricks from the Q&A session. 

What were the criteria for curating the lectin panel for Glysite Scout kits?  

The selection process focused on identifying a broad set of biologically relevant lectins for different research fields. An initial literature search identified lectins and glycans most frequently investigated across various fields. Then, key opinion leaders offered input on the selected panel and recommended the inclusion or removal of specific lectins in the kits. 

Is it possible to customize a Glysite Scout kit if it doesn’t contain a lectin of interest? 

Although the curation process focused on creating a panel with a broad selection of lectins, Glysite Scout kits are not comprehensive and might miss a lectin of interest. In those cases, you can customize the lectin panel to fit your experimental needs. In addition to the curated lectin panel, Glysite Scout kits provide all the necessary reagents—ancillary reagents, blocking solutions, and optimized protocols—for a plug-and-play workflow. After you identify the ideal lectin for your application, you can perform your experiment using the new lectin and the reagents provided in the kit while following the accompanying optimized protocol.  

Are Glysite Scout kits recommended for tissues and techniques other than the ones tested and optimized by Vector Laboratories? 

Glysite Scout kits have been tested and optimized for fluorescent detection of glycans in many human and mouse tissue types: Colon, lung, spleen, kidney, liver, pancreas, testes, heart, bladder, and uterus. However, lectins are versatile tools and can be used in other tissue types and applications. For example, flow cytometry experiments frequently use lectin staining. Although Glysite Scout kits haven’t been optimized for this application, they can yield high-quality results if you add a few extra steps when planning your experiment.  

A literature review can help you identify previous studies that used lectin staining in applications like those you intend to use. Then, optimizing your protocol based on published methodologies and results is essential. You can also reach out to the Vector Laboratories’ technical support team, who will help you identify the ideal lectin for a specific application or tissue type.  

How can someone verify the specificity of lectin staining in a new tissue sample or application?  

Running a few extra steps can help you validate your results in a different tissue or system that Vector Laboratories hasn’t previously tested. Pre-incubation with an inhibiting sugar neutralizes the glycan-binding ability of lectins. Alternatively, you can treat the tissue sample with an enzyme that might remove the glycan of interest. For example, researchers commonly use PNGase F to remove N-glycans. Without the glycan of interest, incubation with lectins will not yield a fluorescent signal. These simple steps can help you ensure the specificity of the lectin staining.  

What are the ideal controls for glycan screening experiments? 

It’s essential to add negative controls to glycan screening experiments to help you detect non-specific signals and identify their sources. For example, if you omit detection reagents—streptavidin-conjugated fluorophores—from the workflow and observe a fluorescent signal in the sample, autofluorescence is likely the source. In that case, adding Vector® TrueVIEW® Autofluorescence Quenching Kit to the experiment can help you solve the problem. In addition, omitting the lectin of interest can help you identify if non-specific binding of the detection reagents is contributing to the background signal. When that happens, avidin/biotin or streptavidin/biotin blocking kits can neutralize endogenous biotin and help reduce background fluorescent signal.       

Watch the entire on-demand webinar below for more information on glycan screening, particularly how to customize the Glysite Scout kits with additional lectins. Visit our Glycobiology Resources Page to learn more about glycobiology and get insights on how to optimize your workflow.  

November 9, 2022
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