I have a question that I hope that you can help to answer. We are using Perkin Elmer's TSA Plus Cyanine3 system (NEL744001KT) and we have run out of the 1x Plus Amplification Diluent (FP1135). I wonder is there a way to make this diluent ourselves as we need it very soon? Also the diluent itself costs are as expensive as the kit. Is there anyone using this system that has had the same problem of the diluent running out? If so we were wondering if you could tell us how to make it DIY style in the lab?
We have had good success with this kit in the fluorescence system.
Hi Sivaraman,
we never used that kit. But we used TSA as an HDAC Inhibitor, and for dillution we used ethanol (pa). Of course you must not forget adding the same amount of EtOH to your control.
And of course, this is only suitable if you have to add very small amounts of EtOH.
Maybe this helps you,
best, Guido.
Hi Guido, thanks for the reply. I should have been clearer with what TSA stands for- Tyramide Signal Amplification. This actually is used to boost the secondary antibody signals, especially if the antigen is lowly expressed or if you want to use a lower antibody concentration (to save some of your antibodies). TSA kits come in different forms- to boost fluorescence, and DAB method chromogens too.
Amplification Diluent (Regular, not "Plus")
0.1 M Borate 8.5 / 6.18 g boric acid up to 1 liter with Q, pH to 8.5 with NaOH
0.003% H2O2 / 100 ul 30% stabilized hydrogen peroxide per liter (Sigma #H1009)
Amplification Diluent can be stored at 4°C for several months. I suggest that you make the borate buffer stock and then add hydrogen peroxide to a smaller volume (100 ml) for daily use.
Source: http://genepath.med.harvard.edu/~cepko/protocol/mike/T4.html
http://www.perkinelmer.com/Content/MSDSDatabase/MSDS_SAT705A001EA_AU_EN.pdf
Thanks all, however, what's the differences between regular one and plus?
Hi Victor. May i know what's the reagent Q? As in you said up to 1L with Q.
Thanks, Victor! That's very useful. However, your link to Harvard is no longer active. Here is a link to a similar protocol
https://sites.google.com/site/helobdellaprotocols/histology/tsa
Sivaraman, I meant to say milliQ - ultrapure- water.
Great link Juan Carlos.
Can anyone help to answer my TSA question? https://www.researchgate.net/post/Can_I_do_Tyramide_Signal_Amplification_using_a_biotin_secondary
Thanks! Alan
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