[Histonet] EGFR ISH protocol for Ventana

Thu Feb 9 06:02:05 CST 2012

------Original message ----------------------------------------

Hi 

Can someone help me with the EGFR ISH protocol for Ventana?

Thanks!

Mei Zheng, HTL(ASCP) QIHC 

Clinical Supervisor 

Immunohistochemistry Lab. 

Department of Pathology 

Brigham and Women's Hospital 

Boston, MA 02115 

Lab Tel: 617 732-7790

------Reply ----------------------------------------

Hi Mei,

Sorry for the delay.

Yes, I have a successful EGFR / Chromosome 7 ISH protocol for the
Ventana Benchmark XT.  

First, the software that you will need to have installed is called "XT
Dual Color Open Probe".  This protocol will use the UltraView Silver ISH
DNP Detection for EGFR and UltraView Red ISH DIG Detection for
Chromosome 7.  The counterstain is Hematoxylin II with Bluing Reagent.

Before I get to the protocol, I'll fill you in on the EGFR and
Chromosome 7 probes.  They do NOT arrive in a dispenser.  They each
arrive in pre-diluted vials, so you will need to fill a "user-fillable
dispenser".  The pre-diluted vials are mixed together into one
user-fillable dispenser to create a "cocktail".  

Don't forget you will also need a "button kit" for "ISH PROBE" because
the user-fillable dispenser will not have the proper commercial bar
code.

One more thing, UltraView Red ISH DIG Detection cannot be dehydrated in
alcohol.  I usually dry the slides at 65C for an hour before I
coverslip. 

Finally, the following is a successful protocol summary.  

1 Deparaffinization [Selected]

2 Extended [Selected]

3 Warmup Slide to [72 Deg C], and Incubate for 4 Minutes (
Deparaffinization )

4 Cell Conditioning [Selected]

5 CC2 [Selected]

6 CC2 Cycle 1 [Selected]

7 Warmup Slide to [80 Deg C], and Incubate for [12 Minutes] ( Cycle 1 )

8 CC2 Cycle 2 [Selected]

9 Incubate for [12 Minutes] ( Cycle 2 )

10 CC2 Cycle 3 [Selected]

11 Incubate for [12 Minutes] ( Cycle 3 )

12 Enzyme [Selected]

13 Apply One Drop of [ISH-PROTEASE 3] ( Enzyme ), Apply Coverslip, and
Incubate for [32 Minutes]

14 Probe Auto Dispense [Selected]

15 Apply One Drop of [ISH Probe 3] ( ISH Probe ), No Coverslip and
Incubate for 8 Minutes

16 Denature [Selected]

17 Warmup Slide to [80 Deg C], and Incubate for [12 Minutes] (
Denaturation )

18 Hybridization [Selected]

19 Warmup Slide to [44 Deg C], and Incubate for 4 Minutes (
Hybridization )

20 Incubate for [6 Hours] ( Hybridization )

21 Stringency Washes [Selected]

22 Stringency Wash #1 [Selected]

23 Warmup Slide to [72 Deg C], and Incubate for [8 Minutes] ( Stringency
Wash #1 )

24 Stringency Wash #2 [Selected]

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

26 Stringency Wash #3 [Selected]

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

28 Silver Detection [Selected]

29 Apply One Drop of SIL ISH DNP RAB, Apply Coverslip, and Incubate for
[20 Minutes]

30 Apply One Drop of SIL ISH DNP CHRC, and Incubate for [4 Minutes]

31 Red Detection [Selected]

32 Apply One Drop of RED ISH DIG MAB, Apply Coverslip, and Incubate for
[20 Minutes]

33 Apply One Drop of RED ISH DIG FR, and Incubate for [4 Minutes]

34 Counterstain [Selected]

35 Apply One Drop of [HEMATOXYLIN II] ( Counterstain ), Apply Coverslip,
and Incubate for [8 Minutes]

36 Post Counterstain [Selected]

37 Apply One Drop of [BLUING REAGENT] ( Post Counterstain ), Apply
Coverslip, and Incubate for [4 Minutes]

38 Post Run LCS Application [Selected]

Christopher Lanigan

Research Technologist

Molecular Pathology

Cleveland Clinic Foundation

9500 Euclid Avenue L3-127

Cleveland, OH 44195

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