[Histonet] IHC Slides
Having problems with IHC slides not looking the same between runs. Does it have to do with how long and at what temp they are in the oven for? Courtney Pierce IHC Specialist Quintiles Translational RD - Oncology Innovation Navigating the new health 610 Oakmont Lane Westmont, IL 60559 Office: + 630-203-6234 courtney.pie...@quintiles.com clinical | commercial | consulting | capital ** IMPORTANT--PLEASE READ This electronic message, including its attachments, is COMPANY CONFIDENTIAL and may contain PROPRIETARY or LEGALLY PRIVILEGED information. If you are not the intended recipient, you are hereby notified that any use, disclosure, copying, or distribution of this message or any of the information included in it is unauthorized and strictly prohibited. If you have received this message in error, please immediately notify the sender by reply e-mail and permanently delete this message and its attachments, along with any copies thereof. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC Slides
Not really. Time in the oven and temp is usually not an issue as long as the tissue is well fixed/processed/infiltrated. René J. --- On Mon, 3/12/12, Courtney Pierce courtney.pie...@quintiles.com wrote: From: Courtney Pierce courtney.pie...@quintiles.com Subject: [Histonet] IHC Slides To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Date: Monday, March 12, 2012, 3:35 PM Having problems with IHC slides not looking the same between runs. Does it have to do with how long and at what temp they are in the oven for? Courtney Pierce IHC Specialist Quintiles Translational RD - Oncology Innovation Navigating the new health 610 Oakmont Lane Westmont, IL 60559 Office: + 630-203-6234 courtney.pie...@quintiles.com clinical | commercial | consulting | capital ** IMPORTANT--PLEASE READ This electronic message, including its attachments, is COMPANY CONFIDENTIAL and may contain PROPRIETARY or LEGALLY PRIVILEGED information. If you are not the intended recipient, you are hereby notified that any use, disclosure, copying, or distribution of this message or any of the information included in it is unauthorized and strictly prohibited. If you have received this message in error, please immediately notify the sender by reply e-mail and permanently delete this message and its attachments, along with any copies thereof. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC slides
So...I have run down slides and done antigen retrieval on my FFPE slides. They are currently in antigen retrieval that has come to room temperature. I am not going to be able to finish the IHC stains until tomorrow. Will it be better to keep the slides in the antigen retrieval solution at 4 degrees overnight, or put it in buffer at 4 degrees overnight? I really don't want to stay here late. Thanks histo-hotties!! Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC slides
We've held slides post-retrieval in buffer (tris, PBS, etc.) overnight with no problem. Linda A. Sebree University of Wisconsin Hospital Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of sgoe...@mirnarx.com Sent: Wednesday, March 02, 2011 1:24 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC slides So...I have run down slides and done antigen retrieval on my FFPE slides. They are currently in antigen retrieval that has come to room temperature. I am not going to be able to finish the IHC stains until tomorrow. Will it be better to keep the slides in the antigen retrieval solution at 4 degrees overnight, or put it in buffer at 4 degrees overnight? I really don't want to stay here late. Thanks histo-hotties!! Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC slides
You can put them %3 H2O2 for 10 minutes. and then drop primary antibody and incubate at 4 degrees overnight. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC slides
Just hold them in buffer overnight. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of sgoe...@mirnarx.com Sent: Wednesday, March 02, 2011 1:24 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC slides So...I have run down slides and done antigen retrieval on my FFPE slides. They are currently in antigen retrieval that has come to room temperature. I am not going to be able to finish the IHC stains until tomorrow. Will it be better to keep the slides in the antigen retrieval solution at 4 degrees overnight, or put it in buffer at 4 degrees overnight? I really don't want to stay here late. Thanks histo-hotties!! Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC slides
In buffer at 4ºC overnight René J. --- On Wed, 3/2/11, sgoe...@mirnarx.com sgoe...@mirnarx.com wrote: From: sgoe...@mirnarx.com sgoe...@mirnarx.com Subject: [Histonet] IHC slides To: histonet@lists.utsouthwestern.edu Date: Wednesday, March 2, 2011, 2:24 PM So...I have run down slides and done antigen retrieval on my FFPE slides. They are currently in antigen retrieval that has come to room temperature. I am not going to be able to finish the IHC stains until tomorrow. Will it be better to keep the slides in the antigen retrieval solution at 4 degrees overnight, or put it in buffer at 4 degrees overnight? I really don't want to stay here late. Thanks histo-hotties!! Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] ihc slides
Hi Cindy, That too has happened to me and the process that you do to remedy it, works for me too. (take off coverslip properly, re-apply DAB, and you get a reaction.) My experience is that the slides are not shot, as described by another Histonetter. There was definitely something else going on. I work with the Dako too. Is it possible that the Autostainer was not programmed to apply antibody in the crucial zones? Look into that possibility. Good Luck. Dana Settembre University Hospital - UMDNJ Newark, NJ -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cynthia Pyse Sent: Monday, January 24, 2011 7:58 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] ihc slides Happy Monday Histonetters I need to pick the brains of the IHC experts out there in histoland. I run IHC on the Dako autostainer. On one of our runs there was not enough DAB to dispense on all the slides. My tech dehydrated the slides and cover slipped them. We printed the stain log on the run and confirmed that the DAB was not dispensed on three slides. The cover slips were removed and the slide were soaked in xylene to remove the cover slipping media. The slides were rehydrated and DAB was applied. There was still no reaction after 10 minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can anyone out there give me a reason that the slides would not react after the reapplication of the DAB. I have done this before when the DAB solution was short on the run and it worked. I can't figure out why this didn't react. I called tech service at Dako and talked to the supervisor, she agrees with me that the reapplication should have worked. I checked the run log and the only missing step is the DAB. The antibodies with the missing DAB have their own controls on the slides. Any thought out there as to what the problem is. I have had the PM done on the Dako which should resolve the problem according to Dako. There has not been a problem since. This mystery will drive me crazy until I can figure out why. I could use help. Thanks in advance, I appreciate any input. Cindy Cindy Pyse, CLT, HT (ASCP) Laboratory/Histology Supervisor X-Cell Laboratories 716-250-9235 e-mail cp...@x-celllab.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] ihc slides
Cynthia, Hematoxylin will inactivate the peroxidase (link) in the tissues. You must go back and re-do the link step and hopefully the antigenic sites will still accept more streptavidin (or similar) conjugated antibody. Cheer. Greg Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PEC1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385 I find that the harder I work, the more luck I seem to have. - Thomas Jefferson Cynthia Pyse cp...@x-celllab.com 1/24/2011 8:57 AM Happy Monday Histonetters I need to pick the brains of the IHC experts out there in histoland. I run IHC on the Dako autostainer. On one of our runs there was not enough DAB to dispense on all the slides. My tech dehydrated the slides and cover slipped them. We printed the stain log on the run and confirmed that the DAB was not dispensed on three slides. The cover slips were removed and the slide were soaked in xylene to remove the cover slipping media. The slides were rehydrated and DAB was applied. There was still no reaction after 10 minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can anyone out there give me a reason that the slides would not react after the reapplication of the DAB. I have done this before when the DAB solution was short on the run and it worked. I can't figure out why this didn't react. I called tech service at Dako and talked to the supervisor, she agrees with me that the reapplication should have worked. I checked the run log and the only missing step is the DAB. The antibodies with the missing DAB have their own controls on the slides. Any thought out there as to what the problem is. I have had the PM done on the Dako which should resolve the problem according to Dako. There has not been a problem since. This mystery will drive me crazy until I can figure out why. I could use help. Thanks in advance, I appreciate any input. Cindy Cindy Pyse, CLT, HT (ASCP) Laboratory/Histology Supervisor X-Cell Laboratories 716-250-9235 e-mail cp...@x-celllab.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet - Statement of Confidentiality This message (including attachments) may contain confidential or privileged information intended for a specific individual or organization. If you have received this communication in error, please notify the sender immediately. If you are not the intended recipient, you are not authorized to use, disclose, distribute, copy, print or rely on this email, and should promptly delete this email from your entire computer system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] ihc slides
I should add that the DAB step may also have quenched all of the linked peroxidases even before the hematoxilin was added (which would finish off any that remained). Greg Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PEC1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385 I find that the harder I work, the more luck I seem to have. - Thomas Jefferson Cynthia Pyse cp...@x-celllab.com 1/24/2011 8:57 AM Happy Monday Histonetters I need to pick the brains of the IHC experts out there in histoland. I run IHC on the Dako autostainer. On one of our runs there was not enough DAB to dispense on all the slides. My tech dehydrated the slides and cover slipped them. We printed the stain log on the run and confirmed that the DAB was not dispensed on three slides. The cover slips were removed and the slide were soaked in xylene to remove the cover slipping media. The slides were rehydrated and DAB was applied. There was still no reaction after 10 minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can anyone out there give me a reason that the slides would not react after the reapplication of the DAB. I have done this before when the DAB solution was short on the run and it worked. I can't figure out why this didn't react. I called tech service at Dako and talked to the supervisor, she agrees with me that the reapplication should have worked. I checked the run log and the only missing step is the DAB. The antibodies with the missing DAB have their own controls on the slides. Any thought out there as to what the problem is. I have had the PM done on the Dako which should resolve the problem according to Dako. There has not been a problem since. This mystery will drive me crazy until I can figure out why. I could use help. Thanks in advance, I appreciate any input. Cindy Cindy Pyse, CLT, HT (ASCP) Laboratory/Histology Supervisor X-Cell Laboratories 716-250-9235 e-mail cp...@x-celllab.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet - Statement of Confidentiality This message (including attachments) may contain confidential or privileged information intended for a specific individual or organization. If you have received this communication in error, please notify the sender immediately. If you are not the intended recipient, you are not authorized to use, disclose, distribute, copy, print or rely on this email, and should promptly delete this email from your entire computer system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] ihc slides
Hi Cynthia, I agree with Greg, you have to re-do your staining from beginning (from AR step) after removing the hematoxilin. Naira ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC Slides, Hotplate vs Oven
Tony's answer (62 degrees C for 25 mins) is the minimum to ensure section adheres to slide. We all have to stain slides that have been baked longer than 30 mins. However, especially for Her2, longer baking times can result in false negatives or at least diminished staining. The justification for baking longer is an attempt to prevent the section washing off or lifting. We are seeing evidence that section adhesion is not a good tradeoff for diminished staining. Ideally both can be achieved with no tradeoff. Tony or others who may wish to answer: 1. How do you monitor the shortened baking time, especially when multiple racks of slides are cut throughout the day? 2. Assuming charged slides and cell conditioning/antigen retrieval are employed, are you seeing major problems with section adhesion? 3. What if slides are received from another institution for staining, or slides get baked longer than 25 mins? Thanks in advance, Eric Gagnon MLT Histology Laboratory Kingston General Hospital Kingston, Ontario, Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC Slides, Hotplate vs Oven
Eric, Some of my thoughts on your discussion questions: 1. How do you monitor the shortened baking time, especially when multiple racks of slides are cut throughout the day? We have timers set for when each rack goes into the oven. This might be an issue for labs with larger batches (we are a Children's hospital) 2. Assuming charged slides and cell conditioning/antigen retrieval are employed, are you seeing major problems with section adhesion? No, not usually. Problem sections tend to be those that are inadequately fixed ( therefore inadequately processed), brain and bone sections. They may tend to lift. 3. What if slides are received from another institution for staining, or slides get baked longer than 25 mins? Again, it is difficult to know the conditions that the tissues have undergone. In-built controls, if present are invaluable. We use a BondMax immunostainer, so the antigen retrieval temperature and time are better controlled than is possible with the manual procedure (or when I do it!). I believe that incomplete fixation causes more problems for morphology and immunohistochemistry than is commonly appreciated. Fix the fixing and most problems are solved - Hows that for a sweeping statement! Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gagnon, Eric Sent: Thursday, 22 April 2010 5:31 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC Slides, Hotplate vs Oven Tony's answer (62 degrees C for 25 mins) is the minimum to ensure section adheres to slide. We all have to stain slides that have been baked longer than 30 mins. However, especially for Her2, longer baking times can result in false negatives or at least diminished staining. The justification for baking longer is an attempt to prevent the section washing off or lifting. We are seeing evidence that section adhesion is not a good tradeoff for diminished staining. Ideally both can be achieved with no tradeoff. Tony or others who may wish to answer: 1. How do you monitor the shortened baking time, especially when multiple racks of slides are cut throughout the day? 2. Assuming charged slides and cell conditioning/antigen retrieval are employed, are you seeing major problems with section adhesion? 3. What if slides are received from another institution for staining, or slides get baked longer than 25 mins? Thanks in advance, Eric Gagnon MLT Histology Laboratory Kingston General Hospital Kingston, Ontario, Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] IHC Slides, Hotplate vs Oven
I'm curious as to whether or not others put their IHC slides on a hotplate after cutting? If so, at what temperature and for how long? Who only uses an oven - and what temp and for how long? Does anyone use both the hotplate and the oven? Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC Slides, Hotplate vs Oven
We use an oven only at 60c for 20 minutes. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert Sent: Tuesday, April 20, 2010 9:40 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC Slides, Hotplate vs Oven I'm curious as to whether or not others put their IHC slides on a hotplate after cutting? If so, at what temperature and for how long? Who only uses an oven - and what temp and for how long? Does anyone use both the hotplate and the oven? Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC Slides, Hotplate vs Oven
I sometimes put cut slides on a warm plate attached to my water bath. One of the plates is set at 55d and the other at 60d. I leave them till all the paraffin is completely melted. I only do this for cases I'm paranoid about staying on! Linda A. Sebree University of Wisconsin Hospital Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert Sent: Tuesday, April 20, 2010 9:40 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC Slides, Hotplate vs Oven I'm curious as to whether or not others put their IHC slides on a hotplate after cutting? If so, at what temperature and for how long? Who only uses an oven - and what temp and for how long? Does anyone use both the hotplate and the oven? Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] IHC Slides, Hotplate vs Oven
We use an oven (62oC) for 25min only. See: Henwood, A., (2005) Effect of Slide Drying at 80°C on Immunohistochemistry J Histotechnol 28(1):45-46. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert Sent: Wednesday, 21 April 2010 12:40 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] IHC Slides, Hotplate vs Oven I'm curious as to whether or not others put their IHC slides on a hotplate after cutting? If so, at what temperature and for how long? Who only uses an oven - and what temp and for how long? Does anyone use both the hotplate and the oven? Laurie Colbert ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet