lost ISH signal


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

My psychological health and professional reputation depends on this! Please

Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole

tel : (33)561004179  fax :(33)561004001

P.S. here is the last part of protocol if you need the earlier steps just
ask...I haven't finished translating from French

Deshydratation:  without DTT

     EtOH 30% + NH4Ac 0,3 M (4,62 g/200 ml)              2'

     EtOH 60% + NH4Ac 0,3 M                        2'

     EtOH 80% + NH4Ac 0,3 M                        2'

     EtOH 95%   puis  100% x2

 Séchage à l'air.

Film bmax Amersham.      24 h
Develloper 3', fixater 5', wash 2' H2O, rince H2O + photo flo 200 (5 ml
/4l) (Kodak products LX24*) Air-Dry the film.

Dip slides in emulsion photo (Amersham emulsion LM1). Dry slides 24h in RT
in dark then 1-3 weeks  at  +4°C, then using Kodak products LX24 * develop
4' wash , dip in a stop wash (+H2O 0,5% CH3COOH), fix 10' and rince in H2O

Coloration Hemalun « light » dehydration alcohol and Xylene/Entellan
*the photo products are diluted 3,3X (1,2 liter for 4 l total)

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