RE: [Histonet] H&E stain problems

From:Gary Gill

Hello Terry:

Terry's right.  Agitation is essential to good staining.  Dye that remains
in cells and tissues represents the difference between what the dye
solutions put in and the rinses take out.  There is bound dye that remains,
and loosely infiltrated dye among the protein molecules that will remain and
reduce crispness and contrast unless removed by pronounced agitation.  Some
is better than none, and more is better than some.

Some general rinse tips:

*	Keep rinse volume high/deep to minimize dilution
*	Rinse in series of at least 3 baths
*	Dip constantly, totally in-and-out about once per second
*	Keep rinses clean (e.g., change/rotate when last bath in a series
begins to be colored)
*	For acid dyes such as eosin, water will extract dye most, methanol,
ethanol, and isopropanol in decreasing degrees
*	0.5% glacial acetic acid in water can be substituted effectively for
95% ethanol

Rinsing is most effective when the difference in concentration between the
dye in the cells and tissue and the dye in the rinses is greatest (i.e.,
difference in chemical potential).  When the rinses are dirty (i.e., the
concentration of dye in the rinse approaches/equals/exceeds that in the
cells), there is no net movement of dye out of the substrate).

To appreciate the value of rinsing, don't do it on an experimental basis.
Take an extra smear or tissue section directly from eosin into absolute
alcohol.  I've done the same thing for the Pap stain, going directly from EA
into absolute alcohol -- the staining results are unrecognizable.

Gary Gill

-----Original Message-----
From: Marshall Terry Dr,Consultant Histopathologist
Sent: Tuesday, May 18, 2004 9:35 AM
To: Connie McManus; Petia P Stefanova; Megan Kear;
Subject: RE: [Histonet] H&E stain problems

Connie remarks:

"In truth, I prefer my hand stained sections better than when they're
stained automatically."

When I first saw what I call x-y stainers, I thought that we had in this,
something that reproduced hand staining. Not so. What I think is lacking is
the brisk agitation necessary to break down the interface between old and
new solution. I'm not so sure about the rinsing either.

Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path  Consultant
Pathologist  Rotherham General Hospital  South Yorkshire  England

-----Original Message-----
From: Connie McManus []
Sent: 18 May 2004 15:09
To: 'Petia P Stefanova'; 'Megan Kear';
Subject: RE: [Histonet] H&E stain problems

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-----
[] On Behalf Of Petia P
Sent: Monday, May 17, 2004 6:45 AM
To: Megan Kear;
Subject: Re: [Histonet] H&E stain problems


I use Harris's hematoxylin which is also regressive and purchase my
hematoxylin and eosin /alcohol-based/ from 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!

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