[Histonet] unstaining H&E slides

[Monfils, Paul]

I don't have a reference, but I can share my technique. First you have to get the coverslips off, which could take anywhere from a half hour to a couple of days in xylene, depending on how long the slides have been coverslipped.  Once the coverslips are off, a half hour in fresh xylene to make sure all traces of old dried mounting medium are removed.  Then a couple of rinses in absolute ethanol to remove the xylene. 1 minute in each is plenty, even less if you agitate the slide rack constantly.  A similar rinse in 95% alcohol. Then into tap water.  Running tap water will remove the eosin within 5 minutes or so, unless you happen to live in an area where the tap water is neutral or slightly acidic.  In most areas tap water is slightly basic.  A couple of drops of ammonium hydroxide in a staining dish of tap water will remove the eosin much faster, less than 30 seconds.  Then I use acid alcohol (1% HCl in 70% ethanol) to remove the hematoxylin. This can take up to 15 minutes or so to get all the stain out, though most of it will be removed within 5 minutes. Also, you can double the HCl concentration to speed things up.  Then rinse well in tap water, followed by distilled water if your staining technique calls for it, and restain with your method of choice.