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View additional product information for Image-iT™ FX Signal Enhancer ReadyProbes™ Reagent - FAQs (R37107)
10 product FAQs found
click反应在叠氮化物和炔烃类间是非常有选择性的。生物体系不可能有其他副反应。任何非特异性本底都是由于染料和多种细胞组件的非共价结合造成的。在click反应后，Select FX信号增强剂在减少染料非特异性电荷连接方面的作用失效；我们不推荐将其与Click-iT检测试剂一同使用。减少本底干扰的最佳方法是增加BSA洗涤的次数。您应始终在同等的处理和检测条件下做无染料或无click反应对照，以证实本底确系染料而非自发荧光所致。此外，您还可在无EdU 或无-EU，仅含溶剂的对照样本上进行完整的click反应，验证click反应信号的特异性。
•需要进行封闭操作以减少由非特异性抗体结合产生的背景荧光。常用的封闭步骤是加入2-5％牛血清白蛋白（fraction V defatted BSA）溶液。另一种方法是采用5-10％二抗种属来源的血清溶液。例如，使用山羊抗小鼠IgG二抗时，样品可以被5-10％正常山羊血清有效封闭。为了进一步减少背景荧光，可以使用Image-iT FX 信号增强剂作为预封闭步骤，减少由偶联物上的染料和细胞组分之间电荷的相互作用引起的非特异性标记。
•如果您使用二抗，确保抗体的种属与样品不同。例如不要对小鼠组织使用抗小鼠二抗。
•滴定测定抗体浓度，使用能获得充足的信号的最低浓度。
•试试荧光标记的一抗，因为它应会降低背景干扰，但这样做也会降低信号强度。
这种二抗非特异性结合的现象可能是由染料电荷引起的，例如带负电荷的染料被带正电荷的细胞组分吸引。为了阻止该现象，使用Image-iT FX Signal Enhancer（货号I36933和R37107）能够抑制结合物上的染料与细胞组分之间的电荷作用，进而阻断非特异性结合。
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
This is not recommended. The ReadyProbes reagents were developed for imaging applications whereas the Ready Flow reagents were optimized for flow cytometry.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.