[Histonet] H&E stain problems

[Connie McManus]

Wow.  What a lot of interesting comments!!  

I agree with Terry re the agitation.  When I watch the stainer do those
dips (I can program how many, but NOT the briskness), I wonder if you
could even call it agitation.  My hand dips are very brisk.  Also, I
don't bother letting the slides stay in the alcohols for 1 minute or 2,
I give the slides about 20 -30 good brisk dips in each solution, then
the timed rinses & staining.  This has always been far more satisfactory
to me than those sllllooooowwwww dips from the machine.

As for the kind of hematoxylin, someone suggested I throw out the Harris
and do Gills III.  I've tried Gill III before and I much prefer the
Harris.  So it's just a matter of personal preference on that... AND
what your pathologist likes *G*

Everyone having an nice Tuesday??? *g*

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


-----Original Message-----
From: Marshall Terry Dr, Consultant Histopathologist
[mailto:Terry.Marshall <@t> rothgen.nhs.uk] 
Sent: Tuesday, May 18, 2004 7:35 AM
To: Connie McManus; Petia P Stefanova; Megan Kear;
histonet <@t> lists.utsouthwestern.edu
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
        terry.marshall <@t> rothgen.nhs.uk

-----Original Message-----
From: Connie McManus [mailto:convmcm <@t> cc.usu.edu]
Sent: 18 May 2004 15:09
To: 'Petia P Stefanova'; 'Megan Kear'; histonet <@t> lists.utsouthwestern.edu
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-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Petia P
Stefanova
Sent: Monday, May 17, 2004 6:45 AM
To: Megan Kear; histonet <@t> 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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