cresyl violet on emulsion dipped slides

From:Donna Simmons <>


Since you have checked all your solutions, the most likely probelm is 
lipid content in the tissue that is forming a physical barrier to the 
emulsion [holes] and possibly a chemical interaction [haze and 
opacity].  I assume the haze is optical and related to the staining, 
rather than a problem with random silver deposition.  Another thing 
to check is that your xylene substitute is used in adequate volume 
and number of changes over time, and in conjunction with VERY 
complete dehydration.  The slightest amount of water can make stains 
seem 'murky' and emulsion seem 'hazy' in coverslipped sections.  I 
know Hemo-D type substitutes [and possibly others] take longer than 
xylene to adequately clear and are very unforgiving of the slightest 
moisture content.

Its absolutely essential to defat frozen brain [and other high lipid 
content] sections before emulsion dipping!  [see my previous post on 
cresyl staining of thick frozen sections of macaque brain].  Often, 
one can get away without defatting on in situ hybridized sections, 
because all the alcohols in the procedure effectively do the job for 
you.  Thus you don't realize the need for defatting until a problem 
such as yours shows up...  Defat as the last part of the in situ 
procedure or store slides and do it the day before dipping, according 
to your own schedule:

Before dipping in emulsion, defat sections in xylene or other organic 
solvent [10-30 min] followed by 100%, 95%, 70% alcohol to water, and 
air drying.   If done at end of in situ procedure, after the last 
high stringency wash run slides up through ascending alcohols to 
xylene and then back down to water as above.  Do this at least one 
day before dipping, as slides must be absolutely, completely dry 
before coating with emulsion [another source of mystery artefact in 
the autoradiography technique].

>Date: 27 Sep 2000 14:33:19 -0500
>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...

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