High background in FISH with TSA

Enrique Castro ecastro at eucmax.sim.ucm.es
Thu Feb 4 09:44:38 EST 1999

	I am using FISH to detect mRNA in specific cells in a cell culture. I
am using biotin-labelled RNA probes and signal amplification bt TSA
direct (streptavidin-HRP and enzyme-catalyzed fluorophore deposition). I
am experiencing high backgrounds as a cloud of bright spots. I have
played with probe concentration and endogenous HRP inactivation step,
however there are  a lot of additional parameters to vary. Which factor
would be more sensitive to reduce background? I am worried of endogenous
biotin. Would digoxigenin perform better? 

Any ideas are wellcome, I am new to these methods
Thank in advance


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