RE: [Histonet] RE: Uneven H&E Staining

From:"Tony Henwood"

For the pale H&E, has the wax been completely removed in the xylene. If slides are dried upright, then the wax flows down the slide possibly covering the last section, requiring longer in the dewax solution. Secondly, after the differentiation stage, the acid/alcohol again may be collecting at the bottom of the slide especially in the automatic stainers prior to water rinsing (increasing the differentiation time for the lowest section). To differentiate these two causes, is the eosin pale as well? If it is then it might be caused by the incomplete de-waxing.

For the long NBF fixation of placenta, has excess blood been removed from the placenta? Or have you checked the concentration of formalin (see Jaspers (1987) J Histotechnol 10 (4): 263-265 for the method using Schiffs reagent).


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: [] On Behalf Of Karla Arrington
Sent: Wednesday, 19 November 2008 12:34 PM
Subject: [Histonet] RE: Uneven H&E Staining

Recently we have had an issue with uneven H&E staining.  Lately slides that have 
three sections on one slide, mostly GI biopsies, and the last section (bottom of 
slide) are paler than the upper two section.  All cut by the same person and the 
stains were great.  What could be the cause of this?  Also with the quality of 
formalin.  Can formalin be purchased as "bad" when it takes 6 days to fix placenta 
or breasts?  What is the cause? 
Karla Arrington, HT(ASCP)

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