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Alexa Fluor™ 555 Tyramide Reagent, 1 vial, 150 slides - FAQs

View additional product information for Tyramide Conjugates - FAQs (B40951, B40955, B56131, B40954, B40953, B40952, B40958, B40957, B40956)

2 product FAQs found

I used a neuron-specific antibody to label my neurons. I can't get enough signal from my fluorescent dye conjugated primary antibody. What can I do to improve it?

Here are our recommendations:

Use one of our extensive selection of secondary antibodies conjugated to bright, photostable Alexa Fluor dyes. The degree of labeling for each conjugate is 2-8 fluorophores per IgG molecule, with potentially three secondary antibody-binding sites per primary antibody, providing signal amplification of approximately 10-20 fluorophores per primary antibody.
Alternatively, primary antibody labeling can be detected with a biotinylated secondary antibody in conjunction with either a fluorescent streptavidin or a streptavidin bridge followed by a biotinylated reporter such as Qdot biotin. Although processing times increase with additional incubation and endogenous biotin-blocking steps, detection sensitivity also improves as a result of the labeled streptavidin.
For low-abundance targets, signal amplification may be necessary for optimal signal-to-noise ratios. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate reactive fluorophore-labeled tyramide radicals. These short-lived tyramide radicals covalently couple to nearby residues, producing an amplified fluorescent signal localized at the HRP-target interaction site.
For improved detection sensitivity with rapidly bleaching dyes, our SlowFade Diamond or ProLong Diamond antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
Please review this web page for further optimization tips (https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/guide-to-immunocytochemistry.html).

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

I have a very low-abundance antigen. How can I amplify my signal?

A common method for amplifying antibody detection is biotin-streptavidin detection, where a biotinylated secondary antibody is combined with subsequent labeling with a dye-conjugated streptavidin. This will amplify the signal by approximately 2-8 times, but endogenous biotin must be blocked beforehand. Another option is to use tyramide-signal amplification, where a horseradish peroxidase conjugate is used with a dye-labeled tyramide. This will amplify the signal by approximately 10-20 times, but endogenous peroxidase will need to be blocked. A final option may be to use a Qdot nanoparticle antibody or streptavidin conjugate, which can yield a signal as much as 40 times higher than a standard organic dye conjugate, depending on the Qdot color.

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