[Histonet] Working Ventana Probe Protocols (EBER)

[Sebree Linda A]

Hi Marc and Histonetters (especially those using XTs),
 
 
I've been trying to work up VMS's new EBER probe with less than stellar
results.
 
I have some questions about the protocols you sent via Histonet back in
early June.  My questions/protocols are in red.
 
You state the following:
 
Depar 16 min  We can only select "depar" without an incubation
time....does your instrument allow you to select a time?  We are doing
this on an XT, is this an instrument difference?  What instrument are
you running your ISH on....Ultra by chance?
 
Enzyme Protease 2 for 4 min. We are using ISH Protease 2 / 12 mins. as
that was our protocol with the "old" stuff.
 
ISH (EBER) probe 4 min. Same
 
Denature @ 85 degrees for 12 min. Same
 
Hybe 1 hour What temperature are you hybridizing at? 
 
3 stringency washes for 8 minutes each At what temperature?
 
Blue detection for 20 minutes Same
 
Counterstain for 4 min We use Nuclear Fast Red for 8 mins.
 
Marc, are you using HybReady?
 
Anyone else having had some success with the new EBER probe, reagents
and software, feel free to comment.  Of the 3 specimens I've run, known
positive soft tissue, bm bx and cell block, the soft tissue and bm bx
had some positivity with the EBER DNP and U6 DNP but the cell block was
negative with both.  There was also lots of blue smearing and haze over
the slides.  I'm afraid this optimization/validation could end up being
very expensive even with our 3 control tissues picked up together on
single slides.
 
Thanks for everyone's help,
 
Linda
 

Linda A. Sebree 
University of Wisconsin Hospital & Clinics 
IHC/ISH Laboratory 
DB1-223 VAH 
600 Highland Ave. 
Madison, WI 53792 
(608)265-6596