This came up on histonet about a year ago.  Someone pointed out that leaving
the slide exposed to damp air (the only kind we have in Florida) between the
last alcohol and the xylene for more than a second allows the residual
alcohol on the slide to pick up water from the air.
I told my student, who was having clearing problems to go from the alcohol
to the xylene without taking time to wipe the edges of the slide.  He did
so, and the clearing problem disappeared.

Allen A. Smith, Ph.D.
Barry University
School of Graduate Medical Sciences
    Podiatric Medicine and Surgery
Miami Shores, Florida  33161-6695 

-----Original Message-----
From: Deborah Faichney [mailto:d.a.faichney@stir.ac.uk]
Sent: Thursday, October 10, 2002 4:33 AM
To: 'histonet@pathology.swmed.edu'
Subject: clearing problems


I am in need of a bit of advice regarding poor clearing of slides.  Our
Haematoxylin and Eosin stain has worked fine for years, but we are now
experiencing random clearing problems which follow no particular pattern. We
often find that our first rack into clearing xylene will  not clear ( it has
what looks like drops of water all over slide surface) or it causes the
xylene to turn milky/cloudy looking.  The second and subsequent rack/s of
slides can often clear perfectly. Also, in a different scenario, the first
rack can be fine and subsequent racks will not clear.  This can happen at
any point during the week. Some weeks we don't have the problem, others do.
We change the whole series weekly.  It doesn't seem to matter if we change
the solvents on a daily basis either (tried that to help, but it doesn't).
We can clear the slides if they are taken back from the xylene into fresh
ethanol then into fresh xylene, although this doesn't work every time. 

Any suggestions greatly appreciated. 

Debbie Faichney
Institute of Aquaculture
University of Stirling
Scotland
 

Our H&E staining method

*	Xylene						5 min
*	100% Ethanol					2 min
*	Methylated spirit					1.5 min
*	Water wash
*	Mayers Haematoxylin				5 min
*	water wash
*	1 % Acid alcohol (to differentiate if necessary)
*	water wash
*	Scotts tap water substitute				1min
*	water wash
*	8 part 1% Eosin(aq) to 1 part Putts eosin		5 min
*	water wash
*	methylated spirit					30 sec
*	100% Ethanol					2min
*	100% Ethanol					1.5 min
*	Xylene						5 min
*	Xylene (to coverslip from)

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