lost ISH signal
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
Molecular Biology Scientist
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE
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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