Patrick,

Since you had no bone detachment at 70 degrees, but also no staining, I
would start there with an overnight AR at 70 degrees.  Then if that
still doesn't work I'd slowly increase either the time or temperature to
the point where you get staining and still have tissue adherence.  

Just my thoughts,

Linda Sebree, HT(ASCP)
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory
A4/204-3224
600 Highland Ave.
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Leung,
Patrick
Sent: Thursday, January 11, 2007 11:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone adhesion problems after HIER


Hi,

I am having problems with cortical bone detaching from the slide during
the antigen retrieval step. The samples I am staining are paraffin
embedded mouse tibia.

I am currently using Superfrost+ slides. The AR is with citrate buffer
(pH6.1) from Dako performed in a waterbath for 15 minutes. The slides
are baked overnight at 59C prior to deparaffinizing. 

I have tried using a steamer and microwave, however I still ended up
with the same results. In addition Superfrost GOLD slides did not help
in keeping the bone on. I found that performing HIER at 70C for three
hrs as suggested in the archives to be ineffective in exposing my
antigen (although the bone did stay attached). 

Do you have any suggestions?

Thanks,
Patrick




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