[Histonet] Eosin too pink

Molinari, Betsy BMolinari <@t> heart.thi.tmc.edu
Fri Jan 13 11:57:38 CST 2006

I have always stained my slides manually and I also use the same Richard
Allen solutions. But my samples deparaffinize thru Xylenes - 100% (x3)
to 95%. Then rinse in tap water and a rinse in DH2O. The following steps
are all done for one minute:
Tap water rinse
Clarifier (Richard Allan)
Tap rinse
Bluing (Richard Allan)
Tap rinse
95% ETOH
Eosin Y
100% ETOH (agitate the slides well to remove excess eosin)
100% ETOH
100% ETOH
Xylenes x3

Betsy Molinari HT (ASCP)
Texas Heart Institute
Cardiovascular Pathology
6770 Bertner Ave.
Houston,TX 77030
832-355-6812 (fax)

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Parker,
Sent: Friday, January 13, 2006 10:42 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Eosin too pink

 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

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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