[Histonet] H&E stain problems

[Gary Gill]

You may have heard that classical music was composed by dead white
Europeans.  Well, Harris hematoxylin was composed by a dead white American
(physician at Jefferson Hospital in 1904).  Gill hematoxylin was composed by
a live white American.  So if you want to liven things up, go with Gill's!

Gary Gill (one and the same)

PS -- No royalties involved, thanks to bad advice in 1972 from corporate
counsel for Johns Hopkins Medical School.

-----Original Message-----
From: Connie McManus [mailto:convmcm <@t> cc.usu.edu] 
Sent: Tuesday, May 18, 2004 10:41 AM
To: 'Marshall Terry Dr,Consultant Histopathologist'; 'Petia P Stefanova';
'Megan Kear'; histonet <@t> lists.utsouthwestern.edu
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] H&E stain problems


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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