Thanks for the tips, Tamara. We are trying to hybridize to
G-protein-coupled receptors in the mouse brain, and believe that our
conditions are acceptable as we can detect specific signal with
radioactive techniques. Our problem is that we are trying to develop
non-isotopic strategies, and have had absolutely no luck with several dig
protocols. We feel that the basic problem is one of sensitivity in being
able to detect mRNA's that have very low copy numbers per cell. 

John



*************************
 John Hohmann
 Neurobiology and Behavior Program
 Box 356460 OB/GYN
 University of Washington
 Seattle, Wa. 98195
 hohm@u.washington.edu
 PH # 206-543-9970
 FAX # 206-543-3915
*************************

On Fri, 26 Jan 2001, Tamara Howard wrote:

> 
> >I am looking for advice in non-radioactive detection of very low
> >abundance mRNA species in mouse brain. Standard biotin or dig-labeled
> >protocols do not work, and for logistical reasons we do not want to use
> >isotopes (although P-33 does detect these mRNAs). I am thinking of trying
> >either a tyramide amplification strategy, or perhaps an enzyme-linked
> >flourescent approach. Or maybe both together. Any help in this area would
> >be Much appreciated!
> 
> Another alternative would be plain old enzymatic detection - both alk
> phos. and HRP can be used and will give quite a bit of amplification. I've
> also used tyramide, but it can be fussy to start dealing with; most labs
> will already have experience with AP and/or HRP, so using those might
> be easier.
> 
> However, have you considered that the reason you aren't getting signal is
> due to your probe or fixation or any of the other zillion thiings that
> thwart mRNA ISH and not the detection?
> 
> Good luck!
> 
> Tamara
> 
> 
> |--------------------------------------------------|
>  Tamara Howard
>  Department of Cell Biology and Physiology
>  University of New Mexico - Health Sciences Center
>  Albuquerque, NM 87131
>  thoward@unm.edu
> |--------------------------------------------------|
> 
> 
> 
> 
>