in situ hyb: eosinophilic cell-specific background

Ljubica Bogic lubi at halia
Tue Feb 20 13:36:56 EST 1996


 Eosin dye was giving me problems as well. Try and use toluidine blue. 
Are you using parafin or freash tissue ?. What kind of probes are you 
using? Can you use a bit more stringent hybd. conditions since your 
signal is well defined and strong??? Is it?
Did you check homology of your probes to other mRNAs (genes from the 
same family with high homology to your gene of interest.?? 
				Lubi
Lubi at halia.pbrc.hawaii.edu
On 14 Feb 1996, Mike Serfas wrote:

> We've been doing in situs for some time on mouse intestinal sections with
> success, but with similar genes in human or rat sections we have an
> annoying problem:  background which localizes specifically to
> eosinophilic cells in the intestinal lamina propria.  The remainder of
> the tissue hybridizes just fine, but this tends to make "unpublishable"
> looking results!
> 
> I suspect that this may be related to the positive charge that attracts
> eosin dye, and perhaps RNA as well?  We have, however, been using an
> acetic anhydride pretreatment in triethanolamine buffer already.
> 
> Before I try the Literature Lottery, does anyone have any ideas what in
> particular causes (and fixes) this background?
> 
> A more or less accurate copy of the protocol I've been using is at
> http://icarus.uic.edu/~azathoth/official.html
> 
> 



More information about the Methods mailing list