[Histonet] New DNA Probes for Kappa and Lambda
Original Message -- Message: 9 Date: Tue, 14 Feb 2012 16:31:29 -0600 From: "Vickroy, Jim" Subject: [Histonet] New DNA Probes for Kappa and Lambda To: "histonet@lists.utsouthwestern.edu" Message-ID: <24a4826e8ef0964d86bc5317306f58a55fdc5de...@mmc-mail.ad.mhsil.com> Content-Type: text/plain; charset="us-ascii" 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 Detecti
[Histonet] EGFR ISH protocol for Ventana
--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 === Please consider the environment before printing this e-mail Cleveland Clinic is ranked one of the top hospitals in America by U.S.News & World Report (2010). Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] K/L bone marrow
-Original Message- Date: Wed, 18 Jan 2012 08:29:48 -0500 From: Clare Thornton Subject: [Histonet] K/L bone marrow To: "'histonet@lists.utsouthwestern.edu'" Message-ID: Content-Type: text/plain; charset="us-ascii" 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 cthorn...@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 === Please consider the environment before printing this e-mail Cleveland Clinic is ranked one of the top hospitals in America by U.S.News & World Report (2010). Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] EBER ISH
Hi Sheila, Sorry for the delay. Yes, I am familiar with the EBER ISH probes on the Ventana Benchmark Ultra. Yes, the software that you will need to have installed is called "U - ISH OPEN PROBES - CHROMOGENIC v3". This protocol will use the BLUE DETECTION and with a RED STAIN II counterstain. 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 Minutes] 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 Histonetters, 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 actually using it. I know that there were inquiries about the protocols because of 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. Thanks, Sheila KDL Pathology Knoxville, TN Christopher Lanigan Research Technologist Molecular Pathology Cleveland Clinic Foundation 9500 Euclid Avenue L3-127 Cleveland, OH 44195 (216) 445-1443 === Please consider the environment before printing this e-mail Cleveland Clinic is ranked one of the top hospitals in America by U.S.News & World Report (2010). Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
