Why DTT in in situ hybridization???

[Istvan Barna]

Hi all,

This DTT is a puzzle for me too. I am a beginner doing in situ
hybridization on brain slices with riboprobes. The temperature of
hybridization is quite high: 58 Co. The chromogen in BCIP/NBT in
alkaline phosphatase reaction, but this comes much later than the
hybridization itself. The hybridization solution contains 0.01M DTT. The
DTT stock is 1M, and stored at -20 Co. The quality of the in situ was OK
few weeks ago. Now, the background is quite strong bringing to naught
the real evaluation of the in situ signals. I was told to use higher
concentration of DTT (5x), and use always freshly prepared DTT and
certainly not freezy stored. I did not check this proposal yet, but as
Byung-Hoon Kim, I cannot figure out what can be that effect of DTT which
can have so significant influence on the hybridization. Any suggestion
is wellcomed.  

steve


In article <3802C251.5DA7212C at musica.mcgill.ca>, Mano
<mdep at musica.mcgill.ca> wrote:

> Also note that, if you are using your RNA in the presence of RNAGuard or
> other RNase inhibitor, DTT is necessary for the inhibitor to function.  In
> the absence of DTT, the use of an RNase inhibitor is presumably useless.  I
> read this once in Methods in Enzymology (cannot recall the volume#)

Someone else said this as well earlier.  I thought it sounded peculiar
but
do not doubt its truth.  Do you have any idea why it is needed?  It just
seems counter intuitive that a reducing agent is needed for the
inhibitor
to function.

Regards,
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology