[Histonet] Working Ventana Probe Protocols (EBER)

[Mark Tarango]

Hi Linda,

I had simliar trouble before I bought and added the extra HybReady to the
protocol (there is one that comes with the detection kit, but you need two
for good staining).  It will clean up your stain and A LOT and remove those
blue splotches that show up when you don't use it.

Mark

On Mon, Jul 25, 2011 at 9:39 AM, Sebree Linda A <LSebree <@t> uwhealth.org>wrote:

> 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
>
>
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