Re: clearing problems

From:"J. A. Kiernan"

Deborah Faichney wrote:
> I am in need of a bit of advice regarding poor clearing of slides.  
> ... looks like drops of water all over slide surface) or it causes the
> xylene to turn milky/cloudy looking.  

Surely this has to be due to water carried into the
xylene because the last alcohol wasn't 100%. Two
possible causes suggest themselves:
1. Insufficient shaking off of solvents (alcohols)
during the steps of dehydration of the stained
2. Contamination of your supposedly absolute alcohol
with water. Test for this by mixing some with new
xylene. It shouldn't go milky. This fors the basis of
a very simple assay for water in alcohols: 
Hall, J (2001) A simple, rapid method for measuring 
the percentage of water in alcohols used for
dehydrating tissues. Biotech. Histochem. 76(1):41-42.

> The second and subsequent rack/s of
> slides can often clear perfectly. 

Could it be that the xylene now contains enough alcohol
to prevent milkiness when more small amounts of water
are carried over into it?  If this is the case, you
will probably see microscopic droplets of water in
the mounted preparations, even though they look
transparent to the unaided eye.

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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