RE: cresyl violet on emulsion dipped slides

Joe, Pam and Donna: 

Thanks for you input.  Unfortunately, I am not sure what I can do to remedy
this according to your suggestions.  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.  She said that
the water could cause "chemical contamination" or something to that effect.
It's even in their handbook.  ( I figure it's so that I go through more,
faster, but I had to consider this guarantee thing).  Also, you should know
that my slides are very dry prior to dipping.  They are dehydrated through
ethanols and then air dried for at least 3 hours.  And, once dipped, I store
them with a packet of dryrite in the container. 

And Pam,
Thank you too.  For how long would you allow the slides to dry once dipped?
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.  Let me know if you still
think its a drying thing.

By the way, I also dip back to back to get more out of an aliquot of
emulsion and let them dry upright in a wire test tube rack. 

And Donna, 
Thanks for the advice.  I did read your message regarding the Macaque brain
and briefly considered the lipids a problem.  However, my sections are 14 to
16 micrometers thick and so did not think this could be it.  What do you
think?  Don't get me wrong, I am very willing to try the extra xylene and
ethanol steps.   But do you still think I really need it for my sections?  

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


-----Original Message-----
From: Pam Marcum []
Sent: September 28, 2000 2:09 PM
To: Saby, Joseph; Hoffert Cyrla;
Subject: RE: cresyl violet on emulsion dipped slides

I would agree with Joseph and add the humidity in the dark room needs to be
very high while dipping and drying.  We had problems with slides drying to
fast and this also created holes in the emulsion layer.  I dipped slides
back to back to reduce the layer of emulsion on the slides.  Then separated
them and dried them standing up.  I also used a humidifier in the dark room
to help keep the humidity high enough to stabilize the drying time of the
 Pam Marcum

-----Original Message-----
From: Saby, Joseph []
Sent: Thursday, September 28, 2000 6:08 AM
To: '';
Subject: RE: cresyl violet on emulsion dipped slides


Almost everyone who has performed autoradiography has experienced these
problems to some extent.

When I was doing this, the emulsion had to be diluted with water 1:1.
Without the water, the emulsion is too thick to coat the slide evenly, and
especially where there would be water from the tissue the emulsion layer
would tend to be uneven. After I had run too many slides through the
emulsion mix, I started getting some of your problems.

First, I would suggest making sure the emulsion is diluted and well mixed.
If not, you will get thick and thin areas with "holes" in the layer due to
water from the tissue.  I think the pink staining may be from a too thick
emulsion layer. Shake extra water off the tissue and wipe the back of the
slide before dipping in emulsion, and periodically stir your emulsion.

I hope this helps.


Joseph A. Saby, BA, HT(ASCP)
Pfizer Global Research and Development
Drug Safety Evaluation
2800 Plymouth Road
Ann Arbor, MI 48105
Phone: (734)-622-3631
FAX:   (734)-622-3866

-----Original Message-----
Sent: Wednesday, September 27, 2000 3:04 PM
Subject: cresyl violet on emulsion dipped slides

Hi, all

Does anyone have experience with cresyl violet acetate staining of post- in
situ hybridization (on fresh frozen sections) slides that have been dipped
in Kodak emulsion?  A long time back, 2 strange things started to happen.
First, I would sometimes get pink spots here and there on random slides
within and/or between racks.  This is a pretty rare event.  Second, I
consistently get this opacity or haze appearing mainly over the tissue.
This is often accompanied by spaces or holes in the emulsion.  This is not
happening on the back side of the slide.  Has anyone else has come across
these phenomena?  I have already tried changing solutions, stocks, lots and
times.  I use xylene substitute and have tried 2 different brands already.
I have considered all light sensitivity aspects.  I have stopped diluting
the emulsion with water and more...

I would really appreciate any suggestions since this haze is essentially
ruining much of my work.  Thanks in advance!


Cyrla Hoffert, MSc.
AstraZeneca R&D Montreal
7171 Frederick Banting
Ville St. Laurent (Montreal), Quebec
H3S 1Z9

* 514.832.3200 ext. 2122
FAX 514.832.3232

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