Would like to confirm Brett's observation that sometimes the sense probe
produces similar staining as the antisense probe. From the results from our
ISH staining services, we have had quite a few examples where the sense
probe stained the same cell populations as the anti-sense though the
staining intensity was much weaker. In one of the examples the staining was
reduced to background levels by reducing sense probe concentration (1/5th
the conc), the anti-sense probe conc was also reduced a similar amount with
a minor decrease in staining intensity. In one case we had to use
alternative sequences from the gene (we use oligo cocktails that hybridize
to different regions in the gene) to minimize the sense staining. In other
cases we just gave up.
Abizar
www.innogenex.com

-----Original Message-----
From: Connolly, Brett [mailto:brett_connolly@merck.com]
Sent: Friday, January 21, 2000 6:46 AM
To: 'HISTONET'; 'Mike Bromley'
Subject: RE: In situ hybridization probes


Mike,
You have encountered a common problem.  There are a couple of possibilities.
Some believe that sense transcripts are sometimes generated in the cell,
maybe as a regulatory mechanism. Or, your sense probe could be complementary
to another irrelevant gene transcript (or one not yet discovered) in those
particular cells. Do you BLAST the sequences against a database?
Or, your method is not stringent enough.

Things to do for starters:

1. decrease probe concentrations of you S and AS probes.
2. Increase hybridization temperature
3. Increase post-hybe wash stringency.
4. Use probes from a different sequence area of the gene; i.e. 3'UTR
5. Compete with unlabelled probes.

This all assumes that the root of the problem is in the hybridization.
Although DIG is not in mammalian cells may the anti-dig is sticking. Use the
Fab antibody and try decreasing the concentration.

Brett


Brett M. Connolly, Ph.D.
Merck Research Laboratories
Department of Pharmacology
WP26A-3000
PO Box 4
West Point, PA 19486
Ph. 215-652-2501
FAX 215-652-2075
e-mail: brett_connolly@merck.com



> ----------
> From: 	Mike Bromley[SMTP:MBromley@picr.man.ac.uk]
> Sent: 	Thursday, January 20, 2000 6:32 AM
> To: 	'histonet'
> Subject: 	In situ hybridization probes
>
> Dear All,
> I have been staining formalin fixed paraffin embedded tissue sections
> for Epstein-Barr encoded small RNA's (EBER's) using the method of
> Helene Breitschopf from the Boehringer manual: Non-radioactive in situ
> hybridization  application manual 2nd edition.(published also in Acta
> Neurapathol (1992) 84:581-587).
>
> The method uses digoxigenin labelled RNA probes, the anti-sense is
> giving good strong staining in the right cells, unfortunately the
> sense probe is also staining the same cells albeit not as strongly.
>
> In the Boehringer manual p137, it states that hybridization with sense
> probes sometimes gives unexpected hybridization signals.
>
> Has anyone got any ideas about why the sense probe should apparently
> be specifically targetting the right cells?
>
> Mike Bromley
> Histology Department
> Paterson Institute
> Wilmslow Road
> Manchester
> M20 9BX
> UK
>
>