[Histonet] RE: questions
When I worked in clinical labs the order was first the priority cases (biopsies, bone marrows, etc.) then numerically. I remember once two physicians brought small biopsies and were in a hurry for the results, both wanted to be first out and I can still hear myself explaining to them that there could only be ONE first! Andi From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Webb, Dorothy L [dorothy.l.w...@healthpartners.com] Sent: Thursday, August 07, 2014 9:54 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions I have a couple of questions to ask where there is no right or wrong answer, just curious as to the process that other labs use. 1. After processing, how do you determine the order in which to cut and stain the blocks..numerical or priority driven? 2. Do you adhere to the 6-72 hours of fixation for breasts or make certain all breast tissue is fixed for a minimum time of, say, 24 hours but, of course no longer than 72? I appreciate your responses and thanks for your time!! I am retiring at the end of this year and trying to optimize some processes beforehand:). Dorothy Webb, HT (ASCP) Regions Histology Technical Specialist 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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] RE: questions
... and I can still hear myself explaining to them that there could only be ONE first! Yes, DAILY!!! Every time someone wants to change things around so they get theirs first, this is our only defense as to why everyone can't get theirs first. 8 years of college doesn't seem to help here... Tim Morken Supervisor, Histology, Electron Microscopy and Neuromuscular Special Studies UC San Francisco Medical Center San Francisco, CA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Grantham, Andrea L - (algranth) Sent: Thursday, August 07, 2014 10:08 AM To: Webb, Dorothy L; 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] RE: questions When I worked in clinical labs the order was first the priority cases (biopsies, bone marrows, etc.) then numerically. I remember once two physicians brought small biopsies and were in a hurry for the results, both wanted to be first out and I can still hear myself explaining to them that there could only be ONE first! Andi From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Webb, Dorothy L [dorothy.l.w...@healthpartners.com] Sent: Thursday, August 07, 2014 9:54 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions I have a couple of questions to ask where there is no right or wrong answer, just curious as to the process that other labs use. 1. After processing, how do you determine the order in which to cut and stain the blocks..numerical or priority driven? 2. Do you adhere to the 6-72 hours of fixation for breasts or make certain all breast tissue is fixed for a minimum time of, say, 24 hours but, of course no longer than 72? I appreciate your responses and thanks for your time!! I am retiring at the end of this year and trying to optimize some processes beforehand:). Dorothy Webb, HT (ASCP) Regions Histology Technical Specialist 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: questions
Our cases are cut by priority. And we don't process breasts. (children's hospital) Hazel Horn Supervisor of Histology/Autopsy/Transcription Anatomic Pathology Arkansas Children's Hospital 1 Children's Way | Slot 820| Little Rock, AR 72202 501.364.4240 direct | 501.364.1241 fax hor...@archildrens.org archildrens.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Webb, Dorothy L Sent: Thursday, August 07, 2014 11:54 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions I have a couple of questions to ask where there is no right or wrong answer, just curious as to the process that other labs use. 1. After processing, how do you determine the order in which to cut and stain the blocks..numerical or priority driven? 2. Do you adhere to the 6-72 hours of fixation for breasts or make certain all breast tissue is fixed for a minimum time of, say, 24 hours but, of course no longer than 72? I appreciate your responses and thanks for your time!! I am retiring at the end of this year and trying to optimize some processes beforehand:). Dorothy Webb, HT (ASCP) Regions Histology Technical Specialist 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ** The information contained in this message may be privileged and confidential and protected from disclosure. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this 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 notify us immediately by replying to the message and deleting it from your computer. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: questions
Sounds like we are all working at the same place! LOL I hear exactly the same questions.is it something they teach to all pathology residents Martha Ward, MT (ASCP) QIHC Manager Molecular Diagnostics Lab Medical Center Boulevard \ Winston-Salem, NC 27157 p 336.716.2109 \ f 336.716.5890 mw...@wakehealth.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Morken, Timothy Sent: Thursday, August 07, 2014 1:18 PM To: 'Grantham, Andrea L - (algranth)'; Webb, Dorothy L; 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] RE: questions ... and I can still hear myself explaining to them that there could only be ONE first! Yes, DAILY!!! Every time someone wants to change things around so they get theirs first, this is our only defense as to why everyone can't get theirs first. 8 years of college doesn't seem to help here... Tim Morken Supervisor, Histology, Electron Microscopy and Neuromuscular Special Studies UC San Francisco Medical Center San Francisco, CA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Grantham, Andrea L - (algranth) Sent: Thursday, August 07, 2014 10:08 AM To: Webb, Dorothy L; 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] RE: questions When I worked in clinical labs the order was first the priority cases (biopsies, bone marrows, etc.) then numerically. I remember once two physicians brought small biopsies and were in a hurry for the results, both wanted to be first out and I can still hear myself explaining to them that there could only be ONE first! Andi From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Webb, Dorothy L [dorothy.l.w...@healthpartners.com] Sent: Thursday, August 07, 2014 9:54 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions I have a couple of questions to ask where there is no right or wrong answer, just curious as to the process that other labs use. 1. After processing, how do you determine the order in which to cut and stain the blocks..numerical or priority driven? 2. Do you adhere to the 6-72 hours of fixation for breasts or make certain all breast tissue is fixed for a minimum time of, say, 24 hours but, of course no longer than 72? I appreciate your responses and thanks for your time!! I am retiring at the end of this year and trying to optimize some processes beforehand:). Dorothy Webb, HT (ASCP) Regions Histology Technical Specialist 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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 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] RE: questions
We embed and cut and by priority for: 1) bone marrows, 2) biopsies, 3) breast cases 4) surgicals, 5) placentas, 6) autopsies. RUSH cases for kidney, liver and heart take priority over all! Occasionally we will have requests for a specific case to be cut earlier than the usual priority list and will do that as needed. The cases are cut in numerical order for bone marrows and biopsies to make it easier to stain, organize and deliver them. (We don't have to look for them for an hour if they are in order and anything missing is immediately apparent.) Since we have four to five people cutting in the morning for surgicals we cut in numeric order by case and also alpha order again to help with staining, check out and delivery. Depending on the person sectioning faster or slower we may have cases out of numeric order at times on the stainer and at check out. We adhere strictly to the breast times for 6 to 72 hours in formalin as minimum and maximum limits. We would be prefer all breast have overnight fixation and that is not possible at times. We use the rule anything not in a cassette by 3PM cannot be processed overnight for breast. Then we know we have 6 hours based on time from gross to completion of formalin on the processors. The trick was to make them understand it had to be grossed and in a cassette by cut off not just soaking in formalin. We have residents. Pam Marcum -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Webb, Dorothy L Sent: Thursday, August 07, 2014 11:54 AM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions I have a couple of questions to ask where there is no right or wrong answer, just curious as to the process that other labs use. 1. After processing, how do you determine the order in which to cut and stain the blocks..numerical or priority driven? 2. Do you adhere to the 6-72 hours of fixation for breasts or make certain all breast tissue is fixed for a minimum time of, say, 24 hours but, of course no longer than 72? I appreciate your responses and thanks for your time!! I am retiring at the end of this year and trying to optimize some processes beforehand:). Dorothy Webb, HT (ASCP) Regions Histology Technical Specialist 651-254-2962 This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: Questions about IHC in Frozen Sections
I agree with Liz, We usually fix with acetone/ethanol 5-10 min then go right into buffer, but occasionally use 2.0% NBF for some antibodies. Our buffer contains 0.1% Tween and our sections can be anywhere from 8-20um depending on the specific project. I think the 30min in acetone is messing up your morphology. Brett Brett M. Connolly, Ph.D. Principal Scientist, Imaging Dept. Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 brett_conno...@merck.com T- 215-652-2501 F- 215-993-6803 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Elizabeth Chlipala Sent: Thursday, December 05, 2013 5:59 PM To: Lewis, Patrick; 'Histonet@lists.utsouthwestern.edu' Subject: [Histonet] RE: Questions about IHC in Frozen Sections Patrick Here is what we do for frozen IHC, this is based upon methods that I received from Gayle Callis. Cut frozen sections and let air dry - at least 20-30 minutes post the last section cut. If we are going to stain that same day or the following day we leave the slides at room temp (we are pretty dry here in Colorado) but if you have issues with humidity you can store them in a dessicator overnight. If you need to store at -80 then we package the slides in smaller slide boxes and only package enough slides for one run to avoid freeze and thaw artifact. So once the slides have dried we place them in slide boxes and in those slide boxes we add a small or medium nylon tissue bag that contains Silica Gel, 6-16 mesh (indicating) we just staple the nylon bag shut. We then use a food sealer to seal the slide box in one of those food sealing bags (we got ours at Cost Co they have them on sale every once and a while, along with the bags) and then that goes in the -80 for storage. The day before we are going to stain we pull out the sealed slide box from the -80 and let it sit on the counter top until the next morning when we open up and then fix with the best method for the particular IHC that we are going to use it could be 10% NBF or 4% paraformaldehyde or one that Gayle recommended to us - its an ethanol/acetone mixture - the protocol is listed below. 1. Fix for 5 minutes in solution made of 75% Acetone and 25% Absolute Ethyl Alcohol. NOTE: We purchase Absolute Ethyl Alcohol in the small bottles. Both Acetone and Absolute Ethyl Alcohol are both stored in the flammable storage cabinet. 2. Rinse in two buffer changes for at least 2 minutes. 3. Continue with staining protocol. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick Sent: Thursday, December 05, 2013 3:29 PM To: 'Histonet@lists.utsouthwestern.edu' Subject: [Histonet] Questions about IHC in Frozen Sections Hi Everyone. I am trying to troubleshoot my IHC on frozen sections. My sections are human tonsil at 7 uM. On charged Superfrost slides. They are stored at -80 after drying for 1 hour. When I use them for IHC, I take them out of the -80 and let them air dry for 1 hour before placing them in cold acetone for 30 minutes to fix. Question: If I place them directly in H20 or TBST pH 8.0 after fixation, will that cause cell lysis? Should I dry the slides after acetone fixation before washing them? If so, for how long? My problem seems to be that the tissue is getting digested on the slide, I am trying to trouble shoot which step is causing my tissues to disintegrate. So far I have tried thicker sections 10, 15 uM (That made the problem worse, I am consider going back to 4 uM sections) I also Changed the concentration of H2O2 for my H202 block from 3% to 0.3%, (In my next IHC attempt I will try to examine the slide at each step to see if I can see loss of integrity) Also in my next attempt I plan to eliminate any H20 washes and dry the slide post acetone fixation before washing in TBST. Also I plan to decrease the amount of Tween20 in my Wash buffer from 0.2% to 0.02%. Any advice would be helpful. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Questions about IHC on Frozen Sections
Dear Brett, Liz and Patrick, I agree with Brett and Liz having been in contact with them over the years. However if your tissue is of human origin and you want to do CD4 and/or CD8 staining, then the acetone/alcohol fixative should NOT be used. The alcohol will ruin Human CD4 and CD8 antigens but does not harm mouse or rat CD4 or CD8 antigen. I learned this from Dr. Chris van der Loos who is now going to be sorely missed by the immunostaining community. For human CD4 and CD8, the sections should be air dried, then fixed in cold 4C reagent grade acetone for 10 minutes, then air dried to let the acetone evaporate before going into the buffer. I never rinsed my solvent fixed frozen sections in water, and if the buffer is not made correctly, as I learned the hard way, the sections can look horrible. Being a purist, my acetone fixed FS were rinsed in 3 changes of pure buffer before equilibrating with in protocol rinse buffer/0.05% Tween 20. IF YOU DO USE the acetone/alochol fixation, the sections are fixed at RT in this mixture and then go directly into buffer for 3 changes. DO NOT AIR DRY AFTER THIS Acetone/alcohol mixture/fixative. If you fix a long, long time in acetone, you can get lesser staining of the antigen. Another clever trick is doing a double cold acetone fixation of air dried frozen sections. This stabilizes the section so that it stays on the slide better, and doesn't harm the antigens. It would work for murine and human FS. Procedure is: Fix air dry section for 10 min in 4C acetone, remove and air dry section for 10 minutes, then return for fix again for 10 min in 4C acetone, then air dry these sections to evaporate away the acetone approx 10 to 15 minutes, rinse in pure buffer, proceed with staining. Do NOT rinse your solvent fixed ( or air dried, unfixed) frozen sections with water (the enemy!), you want to use buffer to maintain isotonicity and cellular integrity of the solvent fixed FS. At the end of a chromogenic protocol (after the chromogen is developed), you can even rinse with pure buffer, then immerse the stained sections into NBF to post fix the section for 10 minutes, rinse gently with running water and then counter stain with hematoxylin.This is also a van der Loos trick to improve the cellular morphology of the nuclei in solvent fixed FS, and doesn't harm the chromogen. Why do you use TBSTw at pH 8? That pH seems to be a big high for IHC, as the norm tends to be pH 7.6? You can also make up this endogenous peroxidase block that will NOT chew your sections up. Solvent fixed frozen sections do NOT like strong hydrogen peroxide concentrations, and this one worked perfectly for us. It is also a published method. PEROXIDASE BLOCK (0.03% hydrogen peroxide) 5 mls DPBS (Dulbeccos, Sigma), pH 7.4 - 7.4 5 ul 30% hydrogen peroxide 50 ul 10% sodium azide Make up, put in a dropper bottle, and use for 1 week, refrigerate. Discard after 1 week or make up fresh daily. Add to section, incubate for 10 - 15 minutes at RT, rinse well after blocking. If you wish, you can drain off the block, and add new half way through the block if the tissue is particularly bloody. If you think the peroxidase block is still too strong, simply do Alkaline phosphatase methods instead. Always let your unfixed frozen sections just taken from -80C freezer, equilibrate for 20 minutes or more to RT before opening a box as water condensation is the enemy to both antigens and morphology. I am sure I have repeated a great deal of what Liz and Brett presented, but it does drive home some points. Take care Gayle Callis HTL,HT/MT (ASCP) -Original Message- I agree with Liz, We usually fix with acetone/ethanol 5-10 min then go right into buffer, but occasionally use 2.0% NBF for some antibodies. Our buffer contains 0.1% Tween and our sections can be anywhere from 8-20um depending on the specific project. I think the 30min in acetone is messing up your morphology. Brett Brett M. Connolly, Ph.D. Principal Scientist, Imaging Dept. Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 http://lists.utsouthwestern.edu/mailman/listinfo/histonet brett_connolly @t merck.com T- 215-652-2501 F- 215-993-6803 -Original Message- From: http://lists.utsouthwestern.edu/mailman/listinfo/histonet histonet-bounces @t lists.utsouthwestern.edu [mailto: http://lists.utsouthwestern.edu/mailman/listinfo/histonet histonet-bounces @t lists.utsouthwestern.edu] On Behalf Of Elizabeth Chlipala Sent: Thursday, December 05, 2013 5:59 PM To: Lewis, Patrick; ' http://lists.utsouthwestern.edu/mailman/listinfo/histonet Histonet @t lists.utsouthwestern.edu' Subject: [Histonet] RE: Questions about IHC in Frozen Sections Patrick Here is what we do for frozen IHC, this is based upon methods that I received from Gayle Callis. Cut frozen sections and let air dry
[Histonet] RE: Questions about IHC in Frozen Sections
Patrick Here is what we do for frozen IHC, this is based upon methods that I received from Gayle Callis. Cut frozen sections and let air dry - at least 20-30 minutes post the last section cut. If we are going to stain that same day or the following day we leave the slides at room temp (we are pretty dry here in Colorado) but if you have issues with humidity you can store them in a dessicator overnight. If you need to store at -80 then we package the slides in smaller slide boxes and only package enough slides for one run to avoid freeze and thaw artifact. So once the slides have dried we place them in slide boxes and in those slide boxes we add a small or medium nylon tissue bag that contains Silica Gel, 6-16 mesh (indicating) we just staple the nylon bag shut. We then use a food sealer to seal the slide box in one of those food sealing bags (we got ours at Cost Co they have them on sale every once and a while, along with the bags) and then that goes in the -80 for storage. The day before we are going to stain we pull out the sealed slide box from the -80 and let it sit on the counter top until the next morning when we open up and then fix with the best method for the particular IHC that we are going to use it could be 10% NBF or 4% paraformaldehyde or one that Gayle recommended to us - its an ethanol/acetone mixture - the protocol is listed below. 1. Fix for 5 minutes in solution made of 75% Acetone and 25% Absolute Ethyl Alcohol. NOTE: We purchase Absolute Ethyl Alcohol in the small bottles. Both Acetone and Absolute Ethyl Alcohol are both stored in the flammable storage cabinet. 2. Rinse in two buffer changes for at least 2 minutes. 3. Continue with staining protocol. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick Sent: Thursday, December 05, 2013 3:29 PM To: 'Histonet@lists.utsouthwestern.edu' Subject: [Histonet] Questions about IHC in Frozen Sections Hi Everyone. I am trying to troubleshoot my IHC on frozen sections. My sections are human tonsil at 7 uM. On charged Superfrost slides. They are stored at -80 after drying for 1 hour. When I use them for IHC, I take them out of the -80 and let them air dry for 1 hour before placing them in cold acetone for 30 minutes to fix. Question: If I place them directly in H20 or TBST pH 8.0 after fixation, will that cause cell lysis? Should I dry the slides after acetone fixation before washing them? If so, for how long? My problem seems to be that the tissue is getting digested on the slide, I am trying to trouble shoot which step is causing my tissues to disintegrate. So far I have tried thicker sections 10, 15 uM (That made the problem worse, I am consider going back to 4 uM sections) I also Changed the concentration of H2O2 for my H202 block from 3% to 0.3%, (In my next IHC attempt I will try to examine the slide at each step to see if I can see loss of integrity) Also in my next attempt I plan to eliminate any H20 washes and dry the slide post acetone fixation before washing in TBST. Also I plan to decrease the amount of Tween20 in my Wash buffer from 0.2% to 0.02%. Any advice would be helpful. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ 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] RE: questions
Hi Dorothy, I have used reagent alcohol for years both with staining and tissue processing. I have never noticed any artifacts or crystals due to use of reagent alcohol. One thing that you should know is that all denatured alcohols are not the same so make sure that you buy a denatured (reagent) alcohol of good quality. As far as keeping slides unstained forever, the one thing that you need to take into consideration is that if the slide is charged or silane coated that the positive charges (adhesiveness) of the slide will change over time and the sections may fall off during staining. Also the antigenicity of some antibodies decreases over time so if you were to do IHC staining, your results may not be as strong as they would be on a more recently cut slide. Best wishes, Debbie Siena HT (ASCP) QIHC Technical Manager | StatLab Medical Products 407 Interchange St. | McKinney, TX 75071 Direct: 972-436-1010 x229 | Fax: 972-436-1369 dsi...@statlab.com | www.statlab.com -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Webb, Dorothy L Sent: Wednesday, February 22, 2012 12:18 PM To: 'histonet@lists.utsouthwestern.edu' Subject: [Histonet] questions For those of you who use reagent alcohol, have you ever experienced any problems in processing or staining, such as artifacts, crystals forming, etc?? How long are unstained slides usable? Do any of you pick up extra sections from a ribbon if the tissue is minimal ? We do and have used them at times, but a pathologist would like them saved forever in case tissue is needed for a molecular test and there is not enough left in the block. Appreciate ahead of time your responses! Dorothy Webb, HT (ASCP) Regions Hospital This e-mail and any files transmitted with it are confidential and are intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient or the individual responsible for delivering the e-mail to the intended recipient, please be advised that you have received this e-mail in error and that any use, dissemination, forwarding, printing, or copying of this e-mail is strictly prohibited. If you have received this communication in error, please return it to the sender immediately and delete the original message and any copy of it from your computer system. If you have any questions concerning this message, please contact the sender. Disclaimer R001.0 ___ 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] RE: Questions
Amy, You do not need to apologize for asking a question to which you did not know the answer. This is an educational avenue, for histology, and there is no such thing as a stupid question if you need answers to solve a problem. Those of us who teach know questions are important, even if you think you know the answer but not exactly sure, or in your case you knew but needed documented verification from others in the field. I hope your fellow workers and supervisors got the message and please feel free to ask. There are those in the field who feel this is a social network for experts and that is okay too, but the real reason NSH and histosearch was started was to expand knowledge of the histology community and to improve our profession. Remembering when histology was in the basement and no one knew we were there, it makes me proud of the progress we have made in the 48 years I have been in the field. Keep asking and share what you know, no need for apologies. Shirley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Senn, Amy R Sent: Monday, September 20, 2010 11:19 AM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Questions I originally asked my questions because I *knew* it was being done incorrectly and no one @ my workplace believed me when I tried to show them the way I was taught/trained-as stated in my original post. Regardless, it never occurred to me that my questions were something that would be met with oh no or oh my gosh - I feel as though I should apologize for my stupid question. However, thank you, to those who responded with your procedures. I'm making a great case based on what we know! Have a good week! Amy R. Senn Holy Spirit Health System 503 N. 21st Street Camp Hill, PA 17011 Phone: 717-763-2124 Fax: 717-763-2947 www.hsh.org Attention: This Message is intended only for the use of 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, you are hereby notified that any dissemination or copying of this message or the taking of any action in reliance on the contents of this message is strictly prohibited. If you have received this message in error, please notify us immediately and destroy the original message. 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
RE: [Histonet] RE: Questions
Agreed! Thanks, Timothy Higgins, HT(ASCP) QIHC Histology Manager APA Amarillo, TX -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Shirley A. Powell Sent: Monday, September 20, 2010 10:33 AM To: Senn, Amy R; Histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Questions Amy, You do not need to apologize for asking a question to which you did not know the answer. This is an educational avenue, for histology, and there is no such thing as a stupid question if you need answers to solve a problem. Those of us who teach know questions are important, even if you think you know the answer but not exactly sure, or in your case you knew but needed documented verification from others in the field. I hope your fellow workers and supervisors got the message and please feel free to ask. There are those in the field who feel this is a social network for experts and that is okay too, but the real reason NSH and histosearch was started was to expand knowledge of the histology community and to improve our profession. Remembering when histology was in the basement and no one knew we were there, it makes me proud of the progress we have made in the 48 years I have been in the field. Keep asking and share what you know, no need for apologies. Shirley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Senn, Amy R Sent: Monday, September 20, 2010 11:19 AM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Questions I originally asked my questions because I *knew* it was being done incorrectly and no one @ my workplace believed me when I tried to show them the way I was taught/trained-as stated in my original post. Regardless, it never occurred to me that my questions were something that would be met with oh no or oh my gosh - I feel as though I should apologize for my stupid question. However, thank you, to those who responded with your procedures. I'm making a great case based on what we know! Have a good week! Amy R. Senn Holy Spirit Health System 503 N. 21st Street Camp Hill, PA 17011 Phone: 717-763-2124 Fax: 717-763-2947 www.hsh.org Attention: This Message is intended only for the use of 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, you are hereby notified that any dissemination or copying of this message or the taking of any action in reliance on the contents of this message is strictly prohibited. If you have received this message in error, please notify us immediately and destroy the original message. 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Questions
Shirley and Amy and others responding to this thread: thanks for your posts. I am not in the histotechnology field - I'm actually a graduate student at Loyola who does her own processing, cutting, and staining of bone tissue - so I really appreciate every so-called dumb question, no matter how simple, since I have never been formally trained in the field. I assumed this was a great forum to safely ask those kinds of questions to advance my graduate student research. I'm learning new details about histology every day just by reading posts by helpful people like you. Thanks have a great week, Kristen Shirley A. Powell powell...@mercer.edu 09/20/10 10:35 AM Amy, You do not need to apologize for asking a question to which you did not know the answer. This is an educational avenue, for histology, and there is no such thing as a stupid question if you need answers to solve a problem. Those of us who teach know questions are important, even if you think you know the answer but not exactly sure, or in your case you knew but needed documented verification from others in the field. I hope your fellow workers and supervisors got the message and please feel free to ask. There are those in the field who feel this is a social network for experts and that is okay too, but the real reason NSH and histosearch was started was to expand knowledge of the histology community and to improve our profession. Remembering when histology was in the basement and no one knew we were there, it makes me proud of the progress we have made in the 48 years I have been in the field. Keep asking and share what you know, no need for apologies. Shirley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Senn, Amy R Sent: Monday, September 20, 2010 11:19 AM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Questions I originally asked my questions because I *knew* it was being done incorrectly and no one @ my workplace believed me when I tried to show them the way I was taught/trained-as stated in my original post. Regardless, it never occurred to me that my questions were something that would be met with oh no or oh my gosh - I feel as though I should apologize for my stupid question. However, thank you, to those who responded with your procedures. I'm making a great case based on what we know! Have a good week! Amy R. Senn Holy Spirit Health System 503 N. 21st Street Camp Hill, PA 17011 Phone: 717-763-2124 Fax: 717-763-2947 www.hsh.org Attention: This Message is intended only for the use of 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, you are hereby notified that any dissemination or copying of this message or the taking of any action in reliance on the contents of this message is strictly prohibited. If you have received this message in error, please notify us immediately and destroy the original message. 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Questions
Well said, Shirley! Debbie M. Boyd, HT(ASCP) l Chief Histologist l Southside Regional Medical Center I 200 Medical Park Boulevard l Petersburg, Va. 23805 l T: 804-765-5050 l F: 804-765-5582 l dkb...@chs.net Shirley A. Powell powell...@mercer.edu Sent by: histonet-boun...@lists.utsouthwestern.edu 09/20/2010 11:37 AM To Senn, Amy R ars...@hsh.org, Histonet@lists.utsouthwestern.edu Histonet@lists.utsouthwestern.edu cc Subject [Histonet] RE: Questions Amy, You do not need to apologize for asking a question to which you did not know the answer. This is an educational avenue, for histology, and there is no such thing as a stupid question if you need answers to solve a problem. Those of us who teach know questions are important, even if you think you know the answer but not exactly sure, or in your case you knew but needed documented verification from others in the field. I hope your fellow workers and supervisors got the message and please feel free to ask. There are those in the field who feel this is a social network for experts and that is okay too, but the real reason NSH and histosearch was started was to expand knowledge of the histology community and to improve our profession. Remembering when histology was in the basement and no one knew we were there, it makes me proud of the progress we have made in the 48 years I have been in the field. Keep asking and share what you know, no need for apologies. Shirley -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Senn, Amy R Sent: Monday, September 20, 2010 11:19 AM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Questions I originally asked my questions because I *knew* it was being done incorrectly and no one @ my workplace believed me when I tried to show them the way I was taught/trained-as stated in my original post. Regardless, it never occurred to me that my questions were something that would be met with oh no or oh my gosh - I feel as though I should apologize for my stupid question. However, thank you, to those who responded with your procedures. I'm making a great case based on what we know! Have a good week! Amy R. Senn Holy Spirit Health System 503 N. 21st Street Camp Hill, PA 17011 Phone: 717-763-2124 Fax: 717-763-2947 www.hsh.org Attention: This Message is intended only for the use of 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, you are hereby notified that any dissemination or copying of this message or the taking of any action in reliance on the contents of this message is strictly prohibited. If you have received this message in error, please notify us immediately and destroy the original message. 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 -- Disclaimer: This electronic message may contain information that is Proprietary, Confidential, or legally privileged or protected. It is intended only for the use of the individual(s) and entity named in the message. If you are not an intended recipient of this message, please notify the sender immediately and delete the material from your computer. Do not deliver, distribute or copy this message and do not disclose its contents or take any action in reliance on the information it contains. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
