More comment on emulsion dipped slides

From:Donna Simmons <>

>I originally used emulsion diluted 1:1
>with water, trying tap water as well as deionized water.  I then stopped
>because I spoke with a Kodak rep who told me not to dilute it, to just use
>it straight.  Otherwise, they cannot guarantee their product.

I consulted with Kodak similarly, and I agree full strength emulsion 
works fine for small, thin, very flat sections and for slides dried 
horizontal.  The problem is that with large-area tissues on slides 
dried vertically the emulsion really must be diluted with the purest 
water available, as Joe says.  Some workers even add a detergent or 
other wetting agent [Kodak Photoflo] to get a very thin even final 
emulsion layer, but I find that not to be essential.  The main 
challenge when diluting emulsion is to mix it thoroughly (I use a 
capped 50ml sterile graduated centrifuge tube and gently turn it 
end-for-end 10 times after the stock emulsion has completely melted), 
and then let it 'rest' at temperature for about half an hour so that 
inevitable bubbles caused by mixing rise to the top and can be 
siphoned off.  These small bubbles can also result in holes in the 
final emulsion layer.
Also, did I understand Joe Saby ["Shake extra water off the tissue 
and wipe the back of the slide before dipping in emulsion] to 
indicate he dips slides wet, out of water?   I guess there are a 
multitude of methods that can work, depending on your tissue, etc.

>I give them about 3 to 4 hours to dry.  Honestly,  I don't think I have a
>humidity problem because I allow my slides to dry in a light safe box,
>normally used for photographic paper.  And when that box is full of dipped
>slides, its pretty humid in there.  Plus, I work in a very small room, sort
>of a large closet to ensure that no one has any reason to enter this
>darkroom while the slides are drying!  With the circulating water bath
>going, I think there is much moisture in the air.

Comments on maintaining adequate humidity while drying over a few 
hours are right on -- something about "eliminating background latent 
image, while allowing the emulsion film to dry without tension so it 
can develop a new latent image from your sample", according to Kodak. 
I've also found that maintaining a constant temperature in the room 
is critical.  It should be on the warm side of normal room temp -- I 
once had a whole set of drying slides ruined by high background 
'tension streaks' in the emulsion layer due to heat shutoff and a 
chilly darkroom over winter holidays!  I guess they assumed nobody 
would be working.. ;-)
Another comment on temperature: accidentally frozen emulsion stock is 
ruined!  If used, slides will have large clumpy background grains and 
any signal will be sporadic.  When ordering in mid-winter, emulsion 
could possibly freeze in air cargo or holding areas in Northern 
cities - another reason to always test a small aliquot of new 
emulsion before dipping valuable slides.

>I briefly considered the lipids a problem.  However, my sections are 14 to
>16 micrometers thick and so did not think this could be it.

I was of the same opinion, until we started having problems similar 
to yours sporadically on 15micron and occasionally on 30micron 
sections.  Adding a solvent defatting step eliminated the problems 
completely.  Also, we've found recently that ending the ISH procedure 
with defatting, followed by 3x100%alc, draining very well and air 
drying for 30min in an air current [hood airflow or deskktop fan, 
protected from dust] saves time and works as well as hydrating to 
water and drying overnight..

>Where I used to work, we used 0.3M ammonium acetate in the ethanols for the
>post-ISH dehydration.  I was once told it helps maintain morphology and
>someone else said its for the drying. 
>Q. Can anyone explain this to me?  Could this be the remedy for my blotching

Acetate salts are used to stabilize DNA hybrids and also some types 
of DAB reaction products.  It could be a leftover from older DNA 
hybridization techniques originally used on blots, or an inclusion 
for in situ techniques using both DAB and radioactive probes in the 
same tissue.
I'd be afraid that the salts would precipitate on the sections and 
cause an artefact, unless they were later washed off with pure water. 
I'm curious to see what others say about this.


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