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Invitrogen™ Image-iT™ FX Signal Enhancer ReadyProbes™ Reagent

Catalog No. R37107
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6 x 2.5 mL kit
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R37107 6 x 2.5 mL kit
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Catalog No. R37107 Supplier Invitrogen™ Supplier No. R37107
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Description

Image-iT™ FX Signal Enhancer ReadyProbes™ Reagent dramatically reduces background fluorescence resulting from non-specific staining during secondary detection using conjugates of streptavidin or secondary antibodies.

Image-iT™ FX Signal Enhancer ReadyProbes™ Reagent dramatically reduces background fluorescence resulting from non-specific staining during secondary detection using conjugates of streptavidin or secondary antibodies. This reagent is applied directly to slides or coverslips containing fixed and permeabilized cell or tissue samples prior to staining with fluorescent probes.

  • Easy protocol—apply ready-made solution using a dropper bottle
  • Compatible with the other steps in your immunostaining protocol
  • Effective with most fluorescent labels
  • Stable at room temperature—keep handy at your scope or cell culture area

Cell imaging applications

Image-iT™ FX Signal Enhancer reagent is a highly effective product for blocking background staining that results from the nonspecific interactions of a wide variety of fluorescent dyes with cell and tissue constituents. Background staining seen with fluorescent conjugates of streptavidin and antibody conjugates are largely eliminated when the Image-iT™ FX Signal Enhancer reagent is applied to fixed and permeabilized cells prior to staining.

Suggestions for use

  • Apply Image-iT™ FX Signal Enhancer reagent directly to slides or coverslips containing cell or tissue samples prior to immune-staining.
  • After the fix/perm step, simply wash and add enough drops to cover your sample, then incubate for 30 min. and proceed with your normal staining protocol.
  • Additional blocking steps may be performed subsequent to blocking with the Image-iT™ Signal Enhancer ReadyProbes™ Reagent, as needed

Specifications

Content And Storage 2.5 mL dropper bottles

Store at ≤25°C.
Product Line Image-iT, ReadyProbes
Product Type Signal Enhancer
Quantity 6 x 2.5 mL kit
Solution Type Signal Enhancer
Format Bottle(s)

Frequently Asked Questions (FAQs)

I used Image-iT FX Signal Enhancer solution to get rid of nonspecific nuclear labeling with Alexa Fluor 568 secondary antibody, but I also saw a significant reduction in my specific mitochondrial antibody labeling. Why is this and what can I do?

The Image-iT FX Signal Enhancer reduces non-specific binding of dye conjugates by blocking positively-charged areas of cells or tissues that attract negatively-charged dyes. In cells after fixation, some positively-charged structures are the nuclei and mitochondria. Thus, you would expect to see a reduction in both mitochondrial and nuclear signal. The lower signal you see afterward is the specific mitochondrial signal; the fluorescence that was lost was the non-specific labeling.
Can I use the Image-iT FX Signal Enhancer instead of my normal blocking solution (BSA or serum)?

No. Image-iT FX Signal Enhancer is not a protein blocker, like BSA, normal serum, or other commercial antibody blockers. Use it as a separate step to block non-specific charge-based binding of dyes to cellular components.
I am labeling fixed and permeabilized cultured cells with an Alexa Fluor secondary antibody. My secondary-only control is showing nuclear and mitochondrial labeling, even though I did a thorough protein blocking and tried a concentration range. What can be done to minimize this non-specific binding?

This is most likely charge-based binding due to interactions of the charge on the dyes with cellular components of opposite charge. This can be blocked by using the Image-iT FX Signal Enhancer Solution which eliminates non-specific binding due to charge. The Signal Enhancer is applied as a pre-blocking step and your regular blocking regimen should be used to limit non-specific binding due to protein-protein interactions. Signal Enhancer cannot be used on live cells.

Can I use the ReadyProbes reagents for flow cytometry?

This is not recommended. The ReadyProbes reagents were developed for imaging applications whereas the Ready Flow reagents were optimized for flow cytometry.
I am observing high background when I analyze my click-labeled samples. What is causing this and what can I do to reduce the background?

The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You can also perform the complete click reaction on a carrier solvent-only, no EdU or no-EU control to verify the specificity of the click reaction signal.

I used a neuron-specific antibody to label my neurons. How can I reduce non-specific antibody binding?

A blocking step should be performed to reduce fluorescence due to non-specific antibody binding. A common blocking step is the addition of a 2-5% solution of bovine serum albumin (fraction V defatted BSA). Another approach employs the addition of a 5-10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti-mouse IgG secondary antibodies, samples may be effectively blocked with 5-10% normal goat serum. To further reduce background fluorescence, the Image-iT FX Signal Enhancer can be included as a pre-blocking step to decrease non-specific labeling due to charge interactions between the dyes on the conjugates and the cellular constituents.
If you are using a secondary antibody make sure that the species of the antibody is not the same as the species of the sample. For example do not use an anti-mouse secondary antibody on mouse tissue.
Titrate the antibody to the lowest concentration you can use and still get adequate signal.
Try using a fluorescently tagged primary antibody because it should give reduced background but be aware this can reduce signal intensity.
I am seeing non-specific binding in the nuclei and mitochondria of my cells with a secondary-only control, but protein binding isn't enough to stop it. What can I do?

What may be happening is non-specific binding of the secondary antibody due to dye charge, for example, where the negatively-charged dye is attracted to positively-charged cellular components. To block this, use Image-iT FX Signal Enhancer (Cat. Nos. I36933 and R37107), which blocks non-specific binding due to charge interactions between the dyes on conjugates and cellular components.

For Research Use Only. Not for use in diagnostic procedures.