[Histonet] H&E water contamination[Scanned]

[Kemlo Rogerson]

How does the water contamination manifest itself? Do you see water droplets
under the scope or do the mounting media just go cloudy? I'm surprised with
the length of time you leave them in the alcohols that you have any
problems, but personally I'd replace the 70% with pure alcohol, as it would
quickly get contaminated with water and I'm not sure that gradations of
alcohol of ascending purity make any difference. I assume the slides are not
cramped into the racks and that there is agitation.

If you use some of the propriety mounting media some are hydroscopic and go
cloudy with time, in my experience. 

Kemlo Rogerson
Cellular Pathology Manager
East Lancashire Hospitals NHS Trust
DD. 01254-294162
Mobile 0774-9754194
 

-----Original Message-----
From: 9msm8 <@t> qlink.queensu.ca [mailto:9msm8 <@t> qlink.queensu.ca] 
Sent: 26 November 2004 22:12
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] H&E water contamination[Scanned]

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
minute).
Any tips would be greatly appreciated,
Mike



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