[Histonet] New DNA Probes for Kappa and Lambda

Wed Feb 15 13:13:26 CST 2012

------------  Original Message  --------------

Message: 9

Date: Tue, 14 Feb 2012 16:31:29 -0600

From: "Vickroy, Jim" <Vickroy.Jim <@t> mhsil.com>

Subject: [Histonet] New DNA Probes for Kappa and Lambda

To: "histonet <@t> lists.utsouthwestern.edu"

      <histonet <@t> lists.utsouthwestern.edu>

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      <24A4826E8EF0964D86BC5317306F58A55FDC5DE697 <@t> mmc-mail.ad.mhsil.com>

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In the past we have used a cocktail DNA probe (ISH) for Kappa and
Lambda.  Has anybody had any experience validating and setting up the
new probes from Ventana which we have to mix the four probes together
and form our own cocktail?  Please share with me your thoughts.   I am
seriously considering sending this test out because of the infrequency,
time, ASR hassle, costs of separate detection kits, etc.

Jim

James Vickroy BS, HT(ASCP)

Surgical  and Autopsy Pathology Technical Supervisor

Memorial Medical Center

217-788-4046

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

Hi Jim

Yes, I have successful Kappa / Lambda ISH protocol for the Ventana
Benchmark XT and also for the Ventana Benchmark ULTRA. 

First, the software that you will need to have installed is called
either "XT ISH Open Probes ChromogenicV3" & "U ISH Open Probes
ChromogenicV3" - depending whether you are running the Benchmark XT or
ULTRA respectively.  These protocols will use the iView BLUE PLUS
DETECTION and with a RED STAIN II counterstain.  I believe each protocol
takes between 6 to 7 hours.

Before I get to the protocols, I'll fill you in on the Kappa and Lambda
probes.  As you know, they do not arrive in a dispenser.  They each
arrive in 4 pre-diluted vials, so you will need to fill a "user-fillable
dispenser".  All four pre-diluted vials are mixed together into one
user-fillable dispenser to make the "cocktail".  Apparently, Ventana is
required to package each separately.

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 the Kappa or Lambda except for the
selected ISH probe.  Therefore, you need 3 slides per case.

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

Finally, the following are successful protocol summaries.  The first is
for the Ventana Benchmark XT, and the second is for the Ventana
Benchmark ULTRA.  

Benchmark XT:

1 Baking [Selected]

2 Warmup Slide to [69 Deg C], and Incubate for [20 Minutes] ( Baking )

3 Deparaffinization [Selected]

4 Standard [Selected]

5 Warmup Slide to [69 Deg C], and Incubate for 4 Minutes (
Deparaffinization )

6 Enzyme [Selected]

7 Apply Coverslip, One Drop of [ISH-PROTEASE 2] ( Enzyme ), and Incubate
for [8 Minutes]

8 Probe [Selected]

9 Probe Auto Dispense [Selected]

10 1 Drop of Probe Dispensed [Selected]

11 Apply One Drop of [ISH Probe 1] ( ISH Probe ), Apply 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 [44 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]

29 Post Run LCS Application [Selected]

Benchmark Ultra:

1 Baking [Selected]

2 Warmup Slide to [69 Deg C], and Incubate for [20 Minutes] ( Baking )

3 Deparaffinization [Selected]

4 Standard [Selected]

5 Warmup Slide to [69 Deg C] from High Temperatures ( Deparaffinization
)

6 Enzyme [Selected]

7 Apply One Drop of [ISH-PROTEASE 2] ( Enzyme ), and Incubate for [8
Minutes]

8 Probe [Selected]

9 Probe Auto Dispense [Selected]

10 1 Drop of Probe Dispensed [Selected]

11 Apply One Drop of [ISH Probe 2] ( 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 [44 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]

29 Post Run LCS Application [Selected]

Chris

Christopher Lanigan

Research Technologist

Molecular Pathology

Cleveland Clinic Foundation

9500 Euclid Avenue L3-127

Cleveland, OH 44195

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