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Wheat Germ Agglutinin, Alexa Fluor™ 594 Conjugate, 5 mg - FAQs

Wheat Germ Agglutinin, Alexa Fluor™ 594 Conjugate, 5 mg - FAQs

View additional product information for Wheat Germ Agglutinin (WGA) - FAQs (W56133, W56132, W56134, W32466, W32465, W11263, W32464, W11262, W11261, W834, W849, W21405, W21404, W7024, Q12021MP, W6748)

7 product FAQs found

My cells are very sensitive and need to be kept in media as much as possible. Is it possible to label the plasma membrane with fluorescent wheat germ agglutinin (WGA) in media instead of buffer?

Yes. Although labeling in buffer (such as Hank's Buffered Saline Solution) is slightly better for brightness and lower non-cell background, media can be used. Do a concentration range to dertermine optimal conditions, since the WGA may potentially bind media components to some extent, slightly decreasing your specific labeling intensity.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can I use CellMask Plasma membrane stains or Alexa Fluor dye labeled wheat germ agglutinin to label the plasma membrane of my paraffin sections?

No. For paraffin sections, there are few options due to the delipidation of the membranes by solvents used in the deparaffinization steps. The only good option is to use an antibody against a plasma membrane protein.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I want to label Golgi bodies in my fixed cells. Is a dye-conjugated lectin like wheat germ agglutinin or concanavalin A a good choice?

No. Those lectins have been shown to label Golgi in fixed and permeabilized cultured cells, but the selectivity is cell type dependent. Usually you wind up with other structures labeling as well, such as endoplasmic reticulum. The only guaranteed way to specifically label Golgi in already fixed cells is to use an antibody for a Golgi-specific antigen.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I injected a fluorescent tracer, but cannot detect it after tissue is fixed and sectioned. What am I doing wrong?

Confirm that the tracer you are using crosslinks to proteins or has a primary amine for fixation-either a hydrazide, lysine fixable dextran, or a protein conjugate.
Use aldehyde-based fixatives to cross link the amines on the tracer.
Inject a larger amount or higher concentration of the tracer. Tracers are generally injected at 1-20% concentrations (10 mg/mL or higher).
Confirm that you are using the correct fluorescent filter for detection. You can perform a spot test by pipetting a small amount of the undiluted stock solution of the tracer onto a slide, then view under the filter you are using on your microscope. This will confirm if the tracer fluorescence can be detected and the fluorescent microscope filter is working properly.
Review tissue fixation and handling procedures to confirm if any reagents or processing procedures could be affecting the tracer.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Do you have a tracer that will only transport retrograde?

Wheat germ agglutinin and cholera toxin conjugates have been used for retrograde tracing. They may have some anterograde tracing in some applications. A selection guide can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing/protein-conjugates.html).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

How do I know which tracer to choose for my experiment?

Factors to consider are size of tracer, method of delivery (injection, direct application to tissue, etc.), and if the tracer needs to be fixable. Here are some links to details about the various classes of neuronal tracers we offer and how to choose between them:

Neuronal Tracing (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html)
Choosing a Tracer (https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorescent-tracers-of-cell-morphology-and-fluid-flow/choosing-a-tracer.html)
Imaging Analysis (http://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/bioprobes-50-journal.pdf)

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I want to label the plasma membrane of my cells, but there are several dyes to choose from. Which one should I use?

For live-cell imaging, the CellVue and CellMask Plasma Membrane Stains are the most uniform and the slowest to be endocytosed. However, they are not the best choice if you wish to fix and permeabilize your cells, such as for antibody labeling. Wheat germ agglutinin (WGA) conjugates are also able to label live cells, or can label already formaldehyde-fixed cells. They can survive subsequent permeabilization with detergents, such as Triton X-100. If cells are already permeabilized, WGA will label internal structures as well. Thus, only an antibody against a plasma membrane protein can be used if cells are already permeabilized. Lipophilic cyanine dyes, such as DiI, will label all cell membranes in live cells, not just plasma membranes, if left on live cells for extended periods. Following page will help you choose (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-structure/plasma-membrane.html).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.