Trouble with emulsion in ISH - emulsion not washing off slides in ISH (Apr/16/2010 )

HI all,

I'm working on a radioactive In Situ Hybridization of sectioned mouse tissue. It's going well except for the final steps of developing and fixing and removing the emulsion coating. I'm hoping someone can offer some insight. I dip the slides in emulsion, let them sit for ~2 weeks and then treat them with developer and fixative where I expect the emulsion to rinse off. However, it is not. Can anyone help me to understand what I might be doing wrong?
Thanks, Kristin

why would you expect the emulsion to wash off? if it does then you lose the silver grains that should show up when you look at the tissue with a microscope. the silver is not released from the emulsion onto the tissue.

mdfenko on Apr 16 2010, 09:24 AM said:

I don't want it to wash off the tissue, but the rest of the slide. It may be that I'm exposing the emulsion to light but that's the type of thing I'm trying to ask about. I have a dark film over my entire slide, not just on the areas of tissue expressing my RNA of interest.

Kristin on Apr 16 2010, 02:09 PM said:

dark emulsion means that it has been exposed to light and/or radiation.

it can also mean that the emulsion is very old. try developing some emulsion on a blank slide. if it comes out dark then you may need to replace the emulsion.

do you use a safelight when working with the emulsion? if so, is it the one recommended by the manufacturer of the emulsion? if not, then how do you work with the emulsion?

mdfenko on Apr 19 2010, 11:35 AM said:

Thanks mdfenko, I actually just work in the dark b/c the red light doesn't give me enough light to make a difference. I'm going to try replacing the emulsion b/c I found out our stock is really old.