[Histonet] EBER ISH

Lanigan, Christopher lanigac <@t> ccf.org
Wed Nov 2 06:19:21 CDT 2011

Hi Sheila,


Sorry for the delay.


Yes, I am familiar with the EBER ISH probes on the Ventana Benchmark

Yes, the software that you will need to have installed is called "U -

This protocol will use the BLUE DETECTION and with a RED STAIN II


Before I get to the protocol, I'll fill you in on the probes.  

They do NOT arrive in a dispenser.  They come in pre-diluted vials, and
you will need to fill a "user-fillable dispense".  

One more thing, you will need to run a control.  This control will
confirm the presence of non-degradated RNA.  The control I used was U6,
and the protocol is identical to EBER except for the selected ISH probe.


By the way, the selected ISH probe will likely be "ISH PROBE 1" or "ISH
PROBE 2" because the user-fillable dispenser will not have the proper
commercial bar code. 


Finally, the following is a successful protocol summary (HybReady Soln
is optional for high background): 


1 Deparaffinization [Selected]

2 Warmup Slide to [69 Deg C], and Incubate for 16 Minutes (
Deparaffinization )

3 Pretreatment [Selected]

4 CC2 [Selected]

5 CC2 Cycle 1 [Selected]

6 CC2 Cycle 2 [Selected]

7 Enzyme [Selected]

8 Apply One Drop of [ISH-PROTEASE 3] ( Enzyme ), and Incubate for [12

9 Probe [Selected]

10 Probe Auto Dispense [Selected]

11 Apply One Drop of [ISH Probe 5] ( ISH Probe ), No Coverslip and
Incubate for 4 Minutes

12 Denature [Selected]

13 Warmup Slide to [75 Deg C], and Incubate for [4 Minutes] (
Denaturation )

14 Hybridization [Selected]

15 Warmup Slide to [50 Deg C], and Incubate for 4 Minutes (
Hybridization )

16 Incubate for [1 Hour] ( Hybridization )

17 Stringency Washes [Selected]

18 Stringency Wash #1 [Selected]

19 Warmup Slide to [60 Deg C], and Incubate for [8 Minutes] ( Stringency
Wash #1 )

20 Stringency Wash #2 [Selected]

21 Incubate for [8 Minutes] ( Stringency Wash #2 )

22 Stringency Wash #3 [Selected]

23 Incubate for [8 Minutes] ( Stringency Wash #3 )

24 Detection Kit [Selected]

25 Blue Detection [Selected]

26 Incubate for [32 Minutes] ( Substrate )

27 Counterstain [Selected]

28 Apply One Drop of [Red Stain II] ( Counterstain ), Apply Coverslip,
and Incubate for [4 Minutes]


Let me know if I forgot anything.


Chris Lanigan






Is anyone out there using the EBER ISH probes from Ventana on the Ultra

Platform?  I have a few questions.  Did you need to get new software

installed?  Was there any training involved?  And do you have successful

protocols that you are willing to share?


I have asked the questions before, but at the time there was nobody

using it.  I know that there were inquiries about the protocols because

the ASR status and the fact that Ventana cannot give  you any advice.


Any and all information would be greatly appreciated since we are
wanting to

start it up at our facility.





KDL Pathology

Knoxville, TN



Christopher Lanigan

Research Technologist

Molecular Pathology

Cleveland Clinic Foundation

9500 Euclid Avenue L3-127

Cleveland, OH 44195

(216) 445-1443



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