Alexa Fluor™ 594 Tyramide SuperBoost™ Kit, streptavidin - FAQs

以下是我们的建议：

•使用我们偶联了明亮的、光稳定性Alexa Fluor 染料的二抗。每个荧光二抗IgG分子上标记有2-8个荧光基团，每个一抗含有三个潜在的二抗结合位点，能够提供大约10-20荧光团/抗体的信号放大等级。
•此外，也可采用生物素结合的二抗以及荧光素-链霉亲和素偶联物检测一抗，或者采用链霉亲和素桥连Qdot 生物素等生物素结合的报告载体来检测一抗。尽管额外的孵育和内源性生物素封闭步骤会增加处理时间，但链霉亲和素标记也会提高检测灵敏度。
•对于低丰度的靶标，为得到最佳信噪比，可能要对信号进行放大。酪胺信号放大技术（TSA）是一种酶介导的检测方法，其利用辣根过氧化物酶（HRP）的催化活性产生具备反应活性的荧光团标记酪胺自由基。这些短寿命的酪胺自由基共价结合到相互作用位点临近的残基上，在HRP-靶相互作用位点上产生放大的荧光信号。
•如需提升快速漂白染料的检测灵敏度，我们的SlowFade Diamond或ProLong Diamond 抗猝灭试剂已经证实可增加光稳定性，降低固定细胞、组织和无细胞制备物中的初始荧光淬灭。
•请访问此网页（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）了解进一步的优化的要点。

放大抗体检测效果的常用方法是生物素-链霉亲和素检测，使生物素化的二抗结合偶联染料的链霉亲和素。该方法能够将信号放大约2-8倍，但在此之前必须先封闭内源性生物素。另一种方法是使用酪胺信号放大技术，将辣根过氧化物酶偶联物与染料标记的酪胺配合使用。该方法能够将信号放大约10-20倍，但在此之前必须先封闭内源性过氧化物酶。最后一种方法是使用Qdot纳米晶体抗体或链霉亲和素偶联物，根据Qdot颜色，该方法可产生高于标准有机染料偶联物40倍的信号。

To limit background, we recommend performing a pre-blocking step with 3% H2O2 for 60 mins to inactivate endogenous peroxidases. To limit the localization of labeling, we recommend optimizing the final concentration of the primary and secondary antibodies and the dye-tyramide. You may also limit the incubation time of the dye-tyramide on the sample.

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

Yes. This involves the sequential application of the antibodies and the tyramides with a HRP-quenching step between antibodies using H2O2.

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The SuperBoost tyramide kits utilize poly-HRP labeled antibodies. This provides a greater number of horseradish peroxidase (HRP) molecules per antibody. The original kits used antibodies and streptavidin that were directly conjugated with HRP and thus, limited the number per antibody or streptavidin.

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

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.

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.