[Histonet] H&E stainng

[Osborn, Sharon]

Helayne,
	Increase your time in the hematoxylin rather than trying to cut the
eosin.  We routinely do 6-8 minutes in hematoxylin.  Most hematoxylins can
stain for up to 15 minutes depending upon the strength of the hematoxylin
and what the pathologist wants.  This is the first step to try.  If you were
using longer times in the hematoxylin and had this problem, I would be
concerned about your tap water washing out the hematoxylin.  In that
situation, it would depend upon the treatment of the tap water (large
chlorine amounts, etc) that creates the difficulty(I encountered this
problem in a hospital lab that had their own well water and self treated
it).
	Again, try increasing your time in the hematoxylin and see if that
solves your problem.  By the way, you live in one of my favorite places,
especially since I have several generations of family there!

sharon osborn
DNAX SP BioPharma
Palo Alto, Ca



Date: Fri, 13 Jan 2006 10:42:11 -0600
From: "Parker, Helayne" <HParker <@t> Skaggs.Net>
Subject: [Histonet] Eosin too pink
To: <histonet <@t> lists.utsouthwestern.edu>
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	<2FABC6145388F24EBA8CBD6EC165BCCB02AFB9E5 <@t> mail1-schc.skaggs.net>
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 Hi List,
  We are currently doing our H & E stain by hand and using the Richard-Allan
7211 Hematoxylin and their Eosin Y.  Our pathologist is still complaining of
slides being TOO PINK.  I have cut the eosin step down to just 5 dips from
15 dips, and still too pink.  The Eosin is very rich. 

This is our protocol:
Hydrate to water like normal (thru Clear Rite 3, 100% and 95% Flex) 
Hematoxylin- 2 mins
Rinsing in water until the water runs clear
Clarifier II- 30 dips
running water -30 dips
Richard Allan Bluing - 30 dips
running water - 30 dips  
95% Flex - 10 dips
Eosin-Y- 5 quick dips
Thru 95%, 100% etc to xylene

Any suggestions on a possible method change or a more mild Eosin. Complaint
seems to solely "TOO PINK".  I have done some experimenting w/ cutting the
eosin w/ 80% Flex- and it does calm it down a bit but, the Hemo looks more
purply then blue, almost periwinkle so not sure if that will be better or
not- we still need to have him review the test slides. Any suggestions ?

Helayne Parker
Histology Section Head
Skaggs Community Health Center
Branson, Missouri


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