[Histonet] RE: Cold plates for icing blocks?
We do when we are sectioning blocks that were processed on the TissueTek Xpress as too much water causes poor staining with these. Jeanine Sanders CDc Atlanta From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Morken, Timothy [timothy.mor...@ucsfmedctr.org] Sent: Monday, November 03, 2014 2:29 PM To: Histonet Subject: [Histonet] Cold plates for icing blocks? Does anyone use a cold plate, like that used for embedding, for icing blocks for sectioning? Just an idea Tim Morken Supervisor, Histology, Electron Microscopy and Neuromuscular Special Studies UC San Francisco Medical Center Box 1656 505 Parnassus Ave San Francisco, CA 94143 USA 415.514-6042 (office) tim.mor...@ucsfmedctr.orgmailto:tim.mor...@ucsfmedctr.org CONFIDENTIALITY NOTICE: This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential, proprietary, and/or privileged information protected by law. If you are not the intended recipient, you may not use, copy, or distribute this email message or its attachments. If you believe you have received this email message in error, please contact the sender by reply email 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: Cold plates for icing blocks?
We use frozen water in Rubbermaid containers. We use the medium size that will last through the cutting of at least 50-60 blocks. As we move blocks off the ice we add uncut blocks. Yes, the ice melts as the morning progresses, but we but gauze on top of the ice to keep the blocks for setting in water. The ice is about an 1 in to 1.5 inches deep so it doesn't melt that fast. Also our room temp is about 67-68 degrees. Debbie M. Boyd HT (ASCP) | Chief Histologist | Southside Regional Medical Center | 200 Medical Park Blvd. | Petersburg, Va. 23805 | PH 804-765-5025 | FAX 804-765-6058 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Sanders, Jeanine (CDC/OID/NCEZID) [j...@cdc.gov] Sent: Thursday, November 06, 2014 8:53 AM To: Morken, Timothy; Histonet Subject: [Histonet] RE: Cold plates for icing blocks? We do when we are sectioning blocks that were processed on the TissueTek Xpress as too much water causes poor staining with these. Jeanine Sanders CDc Atlanta From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] on behalf of Morken, Timothy [timothy.mor...@ucsfmedctr.org] Sent: Monday, November 03, 2014 2:29 PM To: Histonet Subject: [Histonet] Cold plates for icing blocks? Does anyone use a cold plate, like that used for embedding, for icing blocks for sectioning? Just an idea Tim Morken Supervisor, Histology, Electron Microscopy and Neuromuscular Special Studies UC San Francisco Medical Center Box 1656 505 Parnassus Ave San Francisco, CA 94143 USA 415.514-6042 (office) tim.mor...@ucsfmedctr.orgmailto:tim.mor...@ucsfmedctr.org CONFIDENTIALITY NOTICE: This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential, proprietary, and/or privileged information protected by law. If you are not the intended recipient, you may not use, copy, or distribute this email message or its attachments. If you believe you have received this email message in error, please contact the sender by reply email 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 -- 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
[Histonet] HISTO/CYTO SKILL REVIEW
Hi, I was wondering if anyone knows how often Joint Commission requires cytologist and histologist to have their competency reviewed. I'm not talking about their job performance review, but how often to review their job skills with a check list under direct observation. We have been doing it quarterly for histologists. Thanks so much, Marilynn Weber H.T.(ASCP)QIHC Coastal Pathology Consulting Services LLC ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] 2 Sucinyl Cystine stain (AKA 2SC)
Good morning, Does anyone know of a reference lab offering this IHC stain for human FFPE tissue? Thanks, Linda A. Sebree University of Wisconsin Hospital Clinics IHC/ISH Laboratory 600 Highland Ave. Madison, WI 53792 (608)265-6596 FAX: (608)262-7174 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Tissue falling off positive slides
WE are having trouble with tissue coming off our he slides. Our heater is set on 65 degrees for 10 minutes. Any suggestions. We use Fisher blue positive slides. Judith Pardue CHI Memorial Chatt. Tn. 37343 judith_par...@memorial.org This electronic mail and any attached documents are intended solely for the named addressee(s) and contain confidential information. If you are not an addressee, or responsible for delivering this email to an addressee, you have received this email in error and are notified that reading, copying, or disclosing this email is prohibited. If you received this email in error, immediately reply to the sender and delete the message completely from your computer system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Tissue falling off positive slides
Make sure yr sections r airdryed completely before u bake thm Sent from my iPhone On Nov 6, 2014, at 10:34 AM, Pardue, Judith judith_par...@memorial.org wrote: WE are having trouble with tissue coming off our he slides. Our heater is set on 65 degrees for 10 minutes. Any suggestions. We use Fisher blue positive slides. Judith Pardue CHI Memorial Chatt. Tn. 37343 judith_par...@memorial.org This electronic mail and any attached documents are intended solely for the named addressee(s) and contain confidential information. If you are not an addressee, or responsible for delivering this email to an addressee, you have received this email in error and are notified that reading, copying, or disclosing this email is prohibited. If you received this email in error, immediately reply to the sender and delete the message completely from your computer system. ___ 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] Tissue falling off positive slides
Increase the time and/or temperature. I bake mine at 85 Celsius for 20 minutes on non charged slides. Section thickness is also a factor as well as the pH of your blueing reagent. -- Tony Auge HTL (ASCP) QIHC Histology Supervisor - Chandler Pathology Services Cell: (651) 373-4768 Email: tony.a...@gmail.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HELP
Hello All, Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14] (ab2557) for mouse tissue? I have been trying to extinguish the background staining but cannot find the right combination. We are doing this by hand. The recommended dilution for this antibody is 1:50 with HIER. See used protocol below. So far I have tried: 1. concentrations 1:25, 1:50, 1:75, 1:100 - 1:50 gives very high background but good positive staining, 1:100 gives low background but not much positive staining. 2. HIER - 3, 5, 10, 20, minutes @ 96C, 20 minutes at 60C all give the same results - too much back ground 3. We have tried using the H2O2 before the primary and after, increasing the time from 30 minutes to 45 minutes. 4. Tried incubating in the primary for shorter time (20 minutes) and longer overnight at 4C. 5. Tried blocking using 3 different serums, first each one then combinations and longer times. 6. Tried cutting the DAB concentration in 1/2 then 1/3. We have not tried enzyme or acid epitope retrieval yet. *Does anyone have a working protocol or suggestions on what to try?* Thank you in advance. Procedure: Deparaffinize 1. Heat slides to 60C for 10 minutes (oven). 2. Dry slides at room temp 5 minutes. 3. Xylene 5 minutes. 4. Xylene 5 minutes. 5. 100% ethanol 5 minutes (dehydration). 6. 100% ethanol 2 minutes (dehydration). 7. 95% ethanol for 2 minutes (rehydration). 8. 70% ethanol for 2 minutes (rehydration). 9. Run in DI water for 10 minutes. Antigen Retrieval 1. Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes. 2. Cold running water 5 minutes. Staining 1. Wash in PBS for 1 min. 2. Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal bovine serum for 2 hours. 3. Incubate in primary antibody 30-45 minutes at in 2.5% horse serum in PBS. 4. Wash 3x in PBS/tween for 5 minutes each. 5. Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each. 6. Wash 3x PBS 2 minutes each. 7. Incubate with anti-Rat Ig impress solution (according to vector’s instructions) 45 minutes. 8. Wash 3x in PBS for 5 minutes each. 9. Prepare Impact DAB substrate (see insert). 10. Using a light microscope, incubate sections with 50-100ul, until desired darkness occurs (5-30 seconds). 11. Stop DAB reaction using deionized water. 12. Wash in DH2O for 5 minutes 13. Counterstain in Harris hematoxylin for 30 seconds. 14. Wash in DH2O for 5 minutes. 15. Dehydrate 2x in 95% alcohol for 30 seconds each. 16. Dehydrate 3x in 100% alcohol for 2 minutes each. 17. Clear 2x in xylene 5 minutes each. 18. Mount coverslips using Leica mounting media. Hans B Snyder Histologistics 60 Prescott Street Worcester, MA 01605 508-308-7800 h...@histologistics.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] HELP
Hans We use the antibody from Serotec, it works quite nicely. MCA711, proteinase K digestion, rabbit anti-rat secondary and then Envision Rabbit (polymer). We use this antibody at a 1:1200 dilution. 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 March 10, 2014 is Histotechnology Professionals Day 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 Hans B Snyder Sent: Thursday, November 06, 2014 10:31 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] HELP Hello All, Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14] (ab2557) for mouse tissue? I have been trying to extinguish the background staining but cannot find the right combination. We are doing this by hand. The recommended dilution for this antibody is 1:50 with HIER. See used protocol below. So far I have tried: 1. concentrations 1:25, 1:50, 1:75, 1:100 - 1:50 gives very high background but good positive staining, 1:100 gives low background but not much positive staining. 2. HIER - 3, 5, 10, 20, minutes @ 96C, 20 minutes at 60C all give the same results - too much back ground 3. We have tried using the H2O2 before the primary and after, increasing the time from 30 minutes to 45 minutes. 4. Tried incubating in the primary for shorter time (20 minutes) and longer overnight at 4C. 5. Tried blocking using 3 different serums, first each one then combinations and longer times. 6. Tried cutting the DAB concentration in 1/2 then 1/3. We have not tried enzyme or acid epitope retrieval yet. *Does anyone have a working protocol or suggestions on what to try?* Thank you in advance. Procedure: Deparaffinize 1. Heat slides to 60C for 10 minutes (oven). 2. Dry slides at room temp 5 minutes. 3. Xylene 5 minutes. 4. Xylene 5 minutes. 5. 100% ethanol 5 minutes (dehydration). 6. 100% ethanol 2 minutes (dehydration). 7. 95% ethanol for 2 minutes (rehydration). 8. 70% ethanol for 2 minutes (rehydration). 9. Run in DI water for 10 minutes. Antigen Retrieval 1. Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes. 2. Cold running water 5 minutes. Staining 1. Wash in PBS for 1 min. 2. Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal bovine serum for 2 hours. 3. Incubate in primary antibody 30-45 minutes at in 2.5% horse serum in PBS. 4. Wash 3x in PBS/tween for 5 minutes each. 5. Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each. 6. Wash 3x PBS 2 minutes each. 7. Incubate with anti-Rat Ig impress solution (according to vector’s instructions) 45 minutes. 8. Wash 3x in PBS for 5 minutes each. 9. Prepare Impact DAB substrate (see insert). 10. Using a light microscope, incubate sections with 50-100ul, until desired darkness occurs (5-30 seconds). 11. Stop DAB reaction using deionized water. 12. Wash in DH2O for 5 minutes 13. Counterstain in Harris hematoxylin for 30 seconds. 14. Wash in DH2O for 5 minutes. 15. Dehydrate 2x in 95% alcohol for 30 seconds each. 16. Dehydrate 3x in 100% alcohol for 2 minutes each. 17. Clear 2x in xylene 5 minutes each. 18. Mount coverslips using Leica mounting media. Hans B Snyder Histologistics 60 Prescott Street Worcester, MA 01605 508-308-7800 h...@histologistics.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
[Histonet] RE: HISTO/CYTO SKILL REVIEW
We are JCAHO inspected (just this year) and only do yearly competencies. Tom Mc Nemar, HT(ASCP) Histology Supervisor (740) 348-4163 Licking Memorial Hospital 1320 West Main Street Newark, OH 43055 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of rmweber...@comcast.net Sent: Thursday, November 06, 2014 9:56 AM To: histonet Subject: [Histonet] HISTO/CYTO SKILL REVIEW Hi, I was wondering if anyone knows how often Joint Commission requires cytologist and histologist to have their competency reviewed. I'm not talking about their job performance review, but how often to review their job skills with a check list under direct observation. We have been doing it quarterly for histologists. Thanks so much, Marilynn Weber H.T.(ASCP)QIHC Coastal Pathology Consulting Services LLC ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Endogenous Tau (normal Tau)
Hi there, I am writing a poster on different Tau epitopes in human brain free floating sections. I have been unable to get any normal Tau and I wonder what could be the problem. Does anybody know what could be? Also, does anyone have a protocol that can share with me? Thank you! Claudia N. Segovia Senior Neurohistologist Antibody Specialist NeuroScience Associates 10915 Lake Ridge Drive Knoxville, TN 37934 865-675-2245 csego...@nsalabs.com STATEMENT OF CONFIDENTIALITY: The information contained in this electronic message and any attachments are intended for the exclusive use of the addressee(s) and may contain confidential or privileged information. If you are not the intended recipient, or the person responsible for delivering email to the intended recipient, be advised you have received this message in error and any use, dissemination, forwarding, printing or copying is strictly prohibited. Please notify NeuroScience Associates immediately at 865-675-2245 or at csego...@nsalabs.com, and destroy all copies of this message and any attachments. You will be reimbursed for reasonable costs incurred in notifying us. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] RE: HISTO/CYTO SKILL REVIEW
We do a yearly review. We are Joint Commission also. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tom McNemar Sent: Thursday, November 06, 2014 12:41 PM To: 'rmweber...@comcast.net'; histonet Subject: [Histonet] RE: HISTO/CYTO SKILL REVIEW We are JCAHO inspected (just this year) and only do yearly competencies. Tom Mc Nemar, HT(ASCP) Histology Supervisor (740) 348-4163 Licking Memorial Hospital 1320 West Main Street Newark, OH 43055 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of rmweber...@comcast.net Sent: Thursday, November 06, 2014 9:56 AM To: histonet Subject: [Histonet] HISTO/CYTO SKILL REVIEW Hi, I was wondering if anyone knows how often Joint Commission requires cytologist and histologist to have their competency reviewed. I'm not talking about their job performance review, but how often to review their job skills with a check list under direct observation. We have been doing it quarterly for histologists. Thanks so much, Marilynn Weber H.T.(ASCP)QIHC Coastal Pathology Consulting Services LLC ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This e-mail, including attachments, is intended for the sole use of the individual and/or entity to whom it is addressed, and contains information from Licking Memorial Health Systems which is confidential or privileged. If you are not the intended recipient, nor authorized to receive for the intended recipient, be aware that any disclosure, copying, distribution or use of the contents of this e-mail and attachments is prohibited. If you have received this in error, please advise the sender by reply e-mail and delete the message immediately. You may also contact the LMH Process Improvement Center at 740-348-4641. E-mail transmissions cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Thank you. This message, and any included attachments, are from Virtua Health or its related affiliates and is intended only for the addressee(s). The information contained herein is privileged, proprietary or may include confidential information and/or protected patient health information. Any unauthorized review, forwarding, printing, copying, distributing, or otherwise disseminating or taking any action based on such information is strictly prohibited. If you have received this message in error, or have reason to believe you are not authorized to receive it, please delete this message promptly and notify the sender by e-mail with a copy to issecur...@virtua.org. Thank you ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] HELP
We also use the MCA711 antibody (at 1.0 ug/mL, 1 hr.), but with HIER in citrate buffer using a Biocare Decloaker pressure cooker and Biocare's rat-on mouse HRP polymer detection. We get very nice staining with no background. I was never too impressed with ImPRESS. Here are the basics : - Deparaffinize and hydrate to dH20 - Perform HIER, cool for 20 min then wash in H20 - 3.0% H2O2 - 20 min - Serum free block (Biocare Sniper) 30 min - Incubate with MCA771 1 hr. - Incubate with Biocare rat-on-Mouse kit (per instructions) - DAB - 5 min. Washes are with PBS/0.1% Tween Brett Brett M. Connolly, Ph.D. Principle 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, November 06, 2014 12:41 PM To: Hans B Snyder; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] HELP Hans We use the antibody from Serotec, it works quite nicely. MCA711, proteinase K digestion, rabbit anti-rat secondary and then Envision Rabbit (polymer). We use this antibody at a 1:1200 dilution. 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 March 10, 2014 is Histotechnology Professionals Day 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 Hans B Snyder Sent: Thursday, November 06, 2014 10:31 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] HELP Hello All, Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14] (ab2557) for mouse tissue? I have been trying to extinguish the background staining but cannot find the right combination. We are doing this by hand. The recommended dilution for this antibody is 1:50 with HIER. See used protocol below. So far I have tried: 1. concentrations 1:25, 1:50, 1:75, 1:100 - 1:50 gives very high background but good positive staining, 1:100 gives low background but not much positive staining. 2. HIER - 3, 5, 10, 20, minutes @ 96C, 20 minutes at 60C all give the same results - too much back ground 3. We have tried using the H2O2 before the primary and after, increasing the time from 30 minutes to 45 minutes. 4. Tried incubating in the primary for shorter time (20 minutes) and longer overnight at 4C. 5. Tried blocking using 3 different serums, first each one then combinations and longer times. 6. Tried cutting the DAB concentration in 1/2 then 1/3. We have not tried enzyme or acid epitope retrieval yet. *Does anyone have a working protocol or suggestions on what to try?* Thank you in advance. Procedure: Deparaffinize 1. Heat slides to 60C for 10 minutes (oven). 2. Dry slides at room temp 5 minutes. 3. Xylene 5 minutes. 4. Xylene 5 minutes. 5. 100% ethanol 5 minutes (dehydration). 6. 100% ethanol 2 minutes (dehydration). 7. 95% ethanol for 2 minutes (rehydration). 8. 70% ethanol for 2 minutes (rehydration). 9. Run in DI water for 10 minutes. Antigen Retrieval 1. Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes. 2. Cold running water 5 minutes. Staining 1. Wash in PBS for 1 min. 2. Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal bovine serum for 2 hours. 3. Incubate in primary antibody 30-45 minutes at in 2.5% horse serum in PBS. 4. Wash 3x in PBS/tween for 5 minutes each. 5. Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each. 6. Wash 3x PBS 2 minutes each. 7. Incubate with anti-Rat Ig impress solution (according to vector’s instructions) 45 minutes. 8. Wash 3x in PBS for 5 minutes each. 9. Prepare Impact DAB substrate (see insert). 10. Using a light microscope, incubate sections with 50-100ul, until desired darkness occurs (5-30 seconds). 11. Stop DAB reaction using deionized water. 12. Wash in DH2O for 5 minutes 13. Counterstain in Harris hematoxylin for 30 seconds. 14. Wash in DH2O for 5 minutes. 15. Dehydrate 2x in 95% alcohol for 30 seconds each. 16. Dehydrate 3x in 100% alcohol for 2 minutes each. 17. Clear 2x in xylene 5 minutes each. 18. Mount coverslips using Leica mounting media. Hans B Snyder Histologistics 60 Prescott Street Worcester, MA 01605 508-308-7800 h...@histologistics.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One
RE: [Histonet] Tissue falling off positive slides
We bake ours @ 75-80 degrees for 15 minutes in an offline oven, prior to automated staining. Just a habit I picked up years ago, but I always lean the slide racks diagonally against the side of the oven when they're baking, rather than having them sit entirely upright. They seem to drain any residual water more completely that way. We've used Fisher's positive charged slides for years and haven't really had a problem with tissue loss on HE. I think your baking temp and time may be a little low and short, respectively. Good luck. Brian D. Cooper, HT (ASCP)CM | Histology Supervisor Department of Pathology and Laboratory Medicine Children's Hospital Los Angeles 4650 Sunset Blvd MS#43- Los Angeles, CA 90027 Ph: 323.361.3357 Pager: 213-209-0184 bcoo...@chla.usc.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patsy Ruegg Sent: Thursday, November 06, 2014 8:36 AM To: Pardue, Judith Cc: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Tissue falling off positive slides Make sure yr sections r airdryed completely before u bake thm Sent from my iPhone On Nov 6, 2014, at 10:34 AM, Pardue, Judith judith_par...@memorial.org wrote: WE are having trouble with tissue coming off our he slides. Our heater is set on 65 degrees for 10 minutes. Any suggestions. We use Fisher blue positive slides. Judith Pardue CHI Memorial Chatt. Tn. 37343 judith_par...@memorial.org This electronic mail and any attached documents are intended solely for the named addressee(s) and contain confidential information. If you are not an addressee, or responsible for delivering this email to an addressee, you have received this email in error and are notified that reading, copying, or disclosing this email is prohibited. If you received this email in error, immediately reply to the sender and delete the message completely from your computer system. ___ 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 - CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential or legally 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 this original message. - ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Permanent red, Fuchsin and Vector Red not exactly red...
Dear Histonetters, I am trying to combine a peroxidase-DAB staining (brown) with an alkaline phosphatase-staining for detection of two proteins that do not overlap. I have already tried Dako’s Permanent Red and Fuchsin+ and Vector’s Vector Red as AP substrates but I am not getting a clear red color, rather something brownish which is not very different in color from the DAB reaction product. Is there anything I can do to obtain a clearer red color ? Yves ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red...
Yves Try Biocares Warp Red, you will still need to air dry the slides prior to coverslipping do not place them in alcohol, air dry, place in xylene and then mount. 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 March 10, 2014 is Histotechnology Professionals Day 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 Yves Heremans Sent: Thursday, November 06, 2014 1:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red... Dear Histonetters, I am trying to combine a peroxidase-DAB staining (brown) with an alkaline phosphatase-staining for detection of two proteins that do not overlap. I have already tried Dako’s Permanent Red and Fuchsin+ and Vector’s Vector Red as AP substrates but I am not getting a clear red color, rather something brownish which is not very different in color from the DAB reaction product. Is there anything I can do to obtain a clearer red color ? Yves ___ 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] Tissue Processor Validation Procedure
Hi, I am wondering if you guys in histoland will like to share your Tissue Processor Validation Procedure with me. Thanks, Adesuyi == CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may contain confidential and privileged information for the use of the designated recipients named above. If you are not the intended recipient, you are hereby notified that you have received this communication in error and that any review, disclosure, dissemination, distribution, or copying of it or its contents is prohibited. If you have received this communication in error, please notify the sender immediately and destroy all copies of this communication and any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Reichert Jung Autocut Microtome LKB Knifemaker For Glycolmethacrylate Blocks For Sale
Hi Histonetters, After completing a skin/tumor project by Glycolmethacrylate (GMA), I have a Reichert Jung 1150 Autocut microtome, LKB 2078 Histo Knifemaker and 2 boxes of 1 glass strips for sale. Sections for this project were cut at 2 microns and stained with HE. Resolution was definitely better in comparison to the routine paraffin sections. I would like to sell all of these items together. Asking $ 4500; best offers will be considered. Marilyn Johnson Edmonton, AB. Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red...
agree Biocares Warp Red is very strong but still has to avoid alcohol. Patsy Ruegg, HT(ASCP)QIHC Ruegg IHC Consulting 40864 E Arkansas Ave Bennett, CO 80102 H 303-644-4538 C 720-281-5406 prueg...@hotmail.com pru...@ihctech.net From: l...@premierlab.com To: yves.herem...@vub.ac.be; histonet@lists.utsouthwestern.edu Date: Thu, 6 Nov 2014 13:43:09 -0700 Subject: RE: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red... CC: Yves Try Biocares Warp Red, you will still need to air dry the slides prior to coverslipping do not place them in alcohol, air dry, place in xylene and then mount. 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 March 10, 2014 is Histotechnology Professionals Day 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 Yves Heremans Sent: Thursday, November 06, 2014 1:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red... Dear Histonetters, I am trying to combine a peroxidase-DAB staining (brown) with an alkaline phosphatase-staining for detection of two proteins that do not overlap. I have already tried Dako’s Permanent Red and Fuchsin+ and Vector’s Vector Red as AP substrates but I am not getting a clear red color, rather something brownish which is not very different in color from the DAB reaction product. Is there anything I can do to obtain a clearer red color ? Yves ___ 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
