In situ hybridization question

[Phyllis Mann]

Thanks for the reply, Peter.

The emulsion is at 42 degrees C, which is supposed to be the optimal
temperature.  I guess it is possible that with the constant dipping of slides,
the emulsion doesn't stay at that temperature even though I keep the dipping
container in a 42 C water bath.  I'll check that out next time.

I do use dessicant in the slide boxes and I let the boxes come to room
temperature at least 2 hours before I develop.

I had heard that the fix "clears" the slides.  I guess I could leave them in
longer in the fix but most people seem to think that 5 minutes is enough.

Thanks again for your suggestions,

Phyllis

Peter Ashby wrote:

> In article <3B1673B2.240FA422 at tufts.edu>,
>  Phyllis Mann <phyllis.mann at tufts.edu> wrote:
>
> > Hi All,
> >
> > I am doing ISHH using 33P labeled-riboprobes on rat brain tissue and I'm
> > having a problem during the development of the slides.  I dip each slide
> > in NBT2 emulsion (diluted 1:1 in water), let dry in the dark standing up
> > for 3 hours and then store in slide boxes at 4 degrees C.  Approx. 2
> > weeks later I develop.  4 minutes D19 (15C), 10 dips in water (stop,
> > 18-20C), 10 minutes in fixer (18-20C), then 30 seconds of running water,
> > then drying in alcohols.  After I counterstain, I usually have a thin
> > film of emulsion on the slides.  Or what I think is emulsion, it looks
> > milky-white.  I've tried variations on the development and it still the
> > same.    In other labs that I have visited they do it quite similarly
> > but do not have the white film on their slides.   Any suggestions?
>
> Two thoughts come to mind. Do you use silica gel in the slide boxes
> during exposure and do you seal them? The milkiness could be caused by
> water entering during exposure (or light leakage). What temperature is
> your emulsion when you dip the slides? I ask because if the emulsion is
> too cold you could be getting a film which is too thick. Unfortunately I
> can't off the top of my head think of what temp I used to use and my
> thesis is at home. A third thought is that maybe your fix is off.
>
> Peter
>
> --
> Peter Ashby
> Wellcome Trust Biocentre
> University of Dundee
> Dundee, Scotland
> Reverse the spam and remove to email me.

--
Phyllis E. Mann, Ph.D.
Clinical Assistant Professor