[Histonet] lost ISH signal, OK now

[Nancy.Walker <@t> sanofi-synthelabo.com]

Remember this:

Help histonetter!

Can you imagine it's like "now you see it now you don't"! A vanishing act
that's flipping me out!!

The pb appears at the photographic emulsion stage, that is the films are
great. It's our silver grains that once devllopped dissappear with time (ie
1-3 weeks depending on the strength of the signal). We haven't been able to
pinpoint any change to our protocol. And sometimes we don't lose the
signal! Is it VOODOO! That is we've changed from Kodak NBT emulsion to
Amersham LM1, it's not changed the pb. We've had no change in products that
we know of (ie devellopper and fixateur are KODAK LX24 ,  Xylene clearing
with ENTELLAN mounting medium).

I would appreciate a discussion on the actual process that could explain
this loss and might help pinpoint the cause. Or has anybody experienced
this.

My psychological health and professional reputation depends on this! Please
Help.

Thanks for all your suggestions;

Here is what I tried :

1.more thorough washing after fixation step of emulsion devellopment - this
certainly helped but 10 days after developping I was still losing
silvergrains with appearance of brown deposits

2. removing Xylene clearing during coverslipping, that is after ETOH steps,
I airdried before mounting with ENTELLAN - this completely eliminated
silver grain loss!!!

3. I tried replacing the ENTELLAN with EUKITT, but in the absence of xylene
this wasn't necessary, I didn't try an aqueous mounting medium, this
suggestion came after I started my trials.

I put the pictures together from an trial that I did with a moderately
abundant RNA target (1 week exposition), the loss is not very dramatic
probably because of longer washes but imagine with a low-expressing RNA
target. I asked Herb Hagler to post the images at : (
http://pathcuri1.swmed.edu). but I myself was unable to access this adress
today. If anyone is interested I can send a small JPG file.

thanks again,

Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE

nancy.walker <@t> sanofi-synthelabo.com
tel : (33)561004179  fax :(33)561004001