[Histonet] K/L bone marrow
lanigac <@t> ccf.org
Fri Jan 20 13:17:23 CST 2012
Date: Wed, 18 Jan 2012 08:29:48 -0500
From: Clare Thornton <CThornton <@t> dahlchase.com>
Subject: [Histonet] K/L bone marrow
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
<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?
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
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
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 (
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] (
14 Hybridization [Selected]
15 Warmup Slide to [44 Deg C], and Incubate for 4 Minutes (
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]
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