2 replies to this topic

[HeatherH]

Hi,

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!

Heather

[bob1]

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.

Edited by bob1, 31 October 2011 - 02:54 PM.

[HeatherH]

Thanks, Bob!  I'll give it a shot.