We use almost the exact same protocol... we use Surgipath Harris, but we
prepare eosin in-house.  One thing I am amazed at in this protocol is
the length of time in the acid alcohol.  Do you use an autostainer?  We
have a Leica.  The time is set to 1 second in the acid ETOH and
sometimes the sections are almost too differentiated.   I can't imagine
6 seconds in the acid ETOH!!  Even when I do H&E manually, I dip the
slides in and quickly put them in running water.  I must have a very
strong solution, I guess. Hmmmm. Interesting.  In truth, I prefer my
hand stained sections better than when they're stained automatically.

Just wondering and blabbering (hey, it's Tuesday, what do you expect??)

Connie McManus
Utah Veterinary Diagnostics Laboratory
Utah State University
Logan, UT
Phone:  435/797-1891
fax: 435/797-2805


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Petia P
Stefanova
Sent: Monday, May 17, 2004 6:45 AM
To: Megan Kear; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] H&E stain problems

Hi,

I use Harris's hematoxylin which is also regressive and purchase my
hematoxylin and eosin /alcohol-based/ from www.surgipath.com.
I get very good H&E staining with this protocol.

REAGENT                       TIME
Xylene                                3 min.
Xylene                                3 min.
Abs. alc.                              2 min.
Abs. alc.                              2 min
95% alc.                              2 min
80% alc.                              2 min
Wash /tap water/                  30 sec.
Hematoxylin                          8 min.
Wash /tap water/                  2 min.
Acid alcohol                          6 sec.
Wash /tap water/                  2 min.
Wash /tap water/                  5 min
80% alc.                              30 sec.
Eosin                                    15 sec.
95% alc.                              10 sec.
Abs. alc.                              30 sec.
Abs. alc.                              30 sec.
Xylene                                1 min.
Xylene                                1 min.
Xylene                                Exit

Hope it helps!
Petia


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