Photo emulsion signal disappeared

Ivo Rieu irieu at sci.kun.nl
Tue Aug 14 07:21:49 EST 2001


Hi,

I performed an in-situ hybridisation on plant material, and in the days
after processing the emulsion the signal disappeared completely (before
I took photos!)

The method:
The RNA-probe was labeled with 33P. I dipped the slides in emulsion from
Amersham: Hypercoat LM-1. After exposure, the slides were developed in
Kodak D-19, rinsed in stop-solutioin (0.5% acetic acid) and fixed in
Kodak fixer and rinsed with tap-water. Then the slides were put through
an ethanol series to dehydrate the tissue, put in xylene two times and
mounted in a xylene based resin, called Eukitt (Merck).

The problem:
The signal was obvious, against a white background it was visible even
by eye. Under the microscope (bright-field and dark-field) the silver
grains were easily seen. But after the weekend, the signal was gone
almost competely!!! Has anyone seen this before? I was thinking that the
emulsion (containing gelatin, but also some kind of plasticizer) may
have dissolved in the xylene/eukitt, and the silver grains diffused
away?? Or that there is something with the breaking of the light, so the
grains are not visible anymore (with high magnification, maybe, some
light brown spots can be seen, but nothing like the normal black silver
grains) Other suggestions??

Thanks in advance, Ivo Rieu




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