Hi Michael

Been there, done that!!  Your problem is summer!  Humidity in the air(since I am from upstate NY myself, I know full well how humid it gets) is contaminating your ethanol and/or xylene sub with water, and while you don't see it when you coverslip, as time goes on the water remaining in the tissues is causing your eosin to bleed out.  Use fresh ethanol every time and increase your times.  What we did to solve that problem was to go from xylene sub(which is extremely intolerant to water of any kind) to plain old xylenes at the end and use a xylene mountant.  It helped dramatically.....and not to worry, the ugly NY winter will soon be upon you(hehehe.....better you than me)!  If you need any more help let me know!

Noelle Linke
Allergan Pharmaceuticals
Irvine, CA
714-246-5568   

-----Original Message-----
From: Michael Becker [mailto:msadk@nycap.rr.com]
Sent: Thursday, August 08, 2002 4:51 PM
To: HistoNet Server
Subject: Re: Eosin bleeding out


Hello,

Long time since I posted.  Please if anyone out there in HistoLand can tell
me what causes and how can I get rid of Eosin counterstain bleeding out of
frozen tissue sections after they are coverslipped and are drying.  Once in
awhile this is a problem.  I have not changed any solutions or technique or
mounting media.  I use the following protocol:

1.  Rapid Fixx-10 to 15 secs
2.  Tap water rinse x2
3. Gill 3 Hemotoxylin 35-60 seconds depending on age of stain
4. Tap water rinse at the sink until water is clear
5.  Lithium Carbonate Bluing-5 to 7 dips
6. 200 proof Ethanol-10 dips
7. 1% Alcoholic Eosin-2 quick dips
8. 200 proof Ethanol-4 containers-10 dips each
9. 200 proof Ethanol-1 container-1 minute
10.Xylene Substitute-Histosolve for 30 secs to a minute or longer.

Coverslip with Histosolve and a Xylene Substitute mounting media-Histomount

I get all my reagents except Ethanol from Shandon and have been very happy
with staining quality.

Any ideas!  Thanks in advance to all who may respond.

Hope summer is treating you all well.  Have fun.  Be safe.

Sue Becker HTL (ASCP)
c/o Dr. Michael J. Mulvaney
MOHS Micrographic Surgery
Albany, NY 12203

"No one knows noses like MOHS!"