[Histonet] Rookie's manual-prep/H&E protocol

Jack England joeamateur <@t> hotmail.com
Tue Dec 9 17:34:20 CST 2003

Hi Majid,

Here's our protocol:

Fix: Place tissue in 1xDPBS for a few hours at 4 degrees C.
(From what I've read, I'm wary of how well this works, so advice would be 
...I prefer 70%EtOH overnight)

70% EtOH --1hr
95% EtOh --1hr
95% EtOh --1hr
3x 100% EtOH--1 hr each

Clear: Xylene overnight

3 x TBS paraffin, 1 hour each at atmospheric pressure (this is one part I'm 
trying to refine)
Embed, and allow to harden overnight.
Section at 7um and apply to slides; allow to dry overnight on slide warmer.
Xylene 1:		3 min
Xylene 2:		2 min
Xylene/100%EtOH:	2 min
100% EtOH:	2 min
95% EtOH:		2 min
80% EtOH:		1 min
70% EtOH:		1 min
50% EtOH:		1 min
di water: 		3 min (per another thread, I consider water deionized at or 
above 18 megaohm-cm)

Stain in Gill's Hematoxylin 2 for 2-4 min (we're still working on optimizing 
this one)
Running tap water: 	1 min
Tap water:	   	1 min
di water:	   	3-5 min

Counterstain in eosin (2.5%--5% diluted 1:2) with a quick dip.

95% EtOH:		2 min
95% EtOH:		2 min
100% EtOH:	2 min
Xylene/100%EtOH:	2 min
Xylene 2:		2 min
Xylene 1:		3 min

Apply mounting media to slide and cover. Let dry in fume hood for a few 
minutes before examining.

Hope this helps!

Jack England

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