[Histonet] K/L bone marrow

[Lanigan, Christopher]

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

Date: Wed, 18 Jan 2012 08:29:48 -0500

From: Clare Thornton <CThornton <@t> dahlchase.com>

Subject: [Histonet] K/L bone marrow

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

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     <C9D78FFC9D668B4CBEA4405F84697504F819B6F1A9 <@t> iris.dahlchase.net>

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Does anyone have a Kappa/Lambda bone marrow ISH protocol for use on the
Ventana Benchmark XT?

 

thanks!

Clare

 

Clare J. Thornton, HTL(ASCP), QIHC

Assistant Histology Supervisor

Dahl-Chase Diagnostic Services

417 State Street, Suite 540

Bangor, ME 04401

cthornton <@t> dahlchase.com

 

-----Reply-----

 

Hi Clare,

 

Sorry for the delay.

 

Yes, I have a successful Kappa / Lambda ISH protocol for Bone Marrow on
the Ventana Benchmark XT.  

 

First, the software that you will need to have installed is called "XT
ISH Open Probes ChromogenicV3".  This protocol will use the iView BLUE+
DETECTION and with a RED STAIN II counterstain.

 

Before I get to the protocol, I'll fill you in on the Kappa and Lambda
probes.  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.  Apparently, Ventana is required to package each separately.

 

Additionally, 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.  

 

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. 

 

One last thing, I was concerned initially about Bone Marrow adhesion to
the charged slides, so I ran a comparison of 2 leading brands.  The
clear winner was Superfrost Plus distributed by Cardinal Health.

 

Finally, the following is a successful protocol summary.  

 

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]

 

Christopher Lanigan

Research Technologist

Molecular Pathology

Cleveland Clinic Foundation

9500 Euclid Avenue L3-127

Cleveland, OH 44195

 

 

 


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