RE: [Histonet] H&E water contamination

From:Tony Henwood

A few ideas.

Check your xylene for water.
Keep the alcohol and xylene containers covered when not in use. Alcohol will
absorb water from the air (possibly xylene as well - though of this I am not


Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: [] 
Sent: Saturday, 27 November 2004 9:12 AM
Subject: [Histonet] H&E water contamination

Hi histonet
I have been performing H&E stains on sliced parraffin embedded mouse
hippocampal slices. I am consistently getting water contamination in my
slides and have been unable to rectify the problem. My procedure for
staining is: Xylene I for 7.5 minutes; Xylene II for 7.5 minutes; 100%
ethanol I for 2 minutes; 100% ethanol II for 2 minutes; 95% ethanol I and
70% ethanol I for 2 minutes each; Water I for a second; then "H" for 20
minutes; Water II for 10 minutes; "E" for about 13 seconds, Water III for
about 1 minute; 70% ethanol II for 50 seconds; 95% ethanol II for 5
minutes; 100% ethanol III and IV for 5 minutes each and then Xylene III
and IV for 7.5 minutes each.
I beleive the "70% ethanol II" is the key step for removing water after
the staining but increasing the amount of time spent in the solution
hasn't worked yet (though I have yet to leave it in for longer then a
Any tips would be greatly appreciated,

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