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double label fISH using two different Alexa-TSA kits - (Oct/31/2011 )


I am trying to get double label fISH to work using Alexa-488-tyramide to detect the first probe, followed by Alexa-594-tyramide to detect the second.

My general protocol is:
1) hybridization of both probes
2) endogenous peroxidase quenching
3) anti-DIG-POD incubation
4) biotin-TSA/ ABC reaction
5) Alexa-488 TSA
6) peroxidase quenching
7) anti-Fluor-POD incubation
8) biotin-TSA/ ABC reaction
5) Alexa-594 TSA

I am getting many more double-labeled cells than anticipated; I suspect cross-reactivity between the two TSAs. Can anyone shed any light on this? How likely is it that I am not quenching peroxidase sufficiently the second time around (I'm using 0.3% H2O2 for 5'?) Given that either method works in a single-label experiment, is there something else that could be the culprit when combining them for double labeling?

Any insight would be greatly appreciated!



I think that your H2O2 conditions should be fine, but it may pay to titrate this out - use up to 3% and/or a longer time (up to 60 min apparently). You could also add some NaN3 to completely inactivate the HRP.


Thanks, Bob! I'll give it a shot.