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: SPAM-LOW: [Histonet] in situ methodology part 2
Louise,
I have been testing a new ISH kit called RISH from BioCare. It works on any
dig labeled mRna probe. The method is designed for ffpe sections but I have
just adapted it to cryosections as well. I can send you some pictures of
Tunnel and EBV on ffpe sections if you would like to see them. Besides
Roche this is the only kit I know of out right now for ISH. They sell the
kit with their probes, I think they only have EBV, Kappa and Lambda and
maybe CMV. They say they are coming out with hpv probes this spring but I
have not seen them yet. They will also sell the reagents without their
probe for researchers like you and me that use our own probes as long as
they are dig labeled.
Ventana has ISH for HPV but the probe is fitc labeled. In research we
prefer dig labeled probes, at least my investigators do.
Regards,
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pru...@ihctech.net
web site www.ihctech.net
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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of louise
renton
Sent: Monday, January 24, 2011 12:31 AM
To: Histonet
Subject: SPAM-LOW: [Histonet] in situ methodology part 2
Hi all
just as clarification .i need to brush up on my background of ISH - what
new products/methododolgies there might be on the market. The tissues I will
be using will either be rodent pups/embryos or formalin fixed/fresh adult
tissue.
As for the probe - thats a stick point - not enough background information
to decide - but probably oligos.
So...the questions still stand:
- where can i find good on-line material
- Who offers the best application manuals - is Roche still int he market?
best regards
--
Louise Renton
Bone Research Unit
University of the Witwatersrand
Johannesburg
South Africa
+27 11 717 2298 (tel fax)
073 5574456 (emergencies only)
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[Histonet] Histotech Needed Martinez, CA ASAP!
Histotechnician Needed - Martinez, CA We are currently seeking experienced Histotechnician to work in a full-time contract position in Martinez, CA. Monday through Friday 6am- 2:30pm. Great opportunity! Histotech needed to prepare tissue specimens for examination; dehydrates, blocks, cuts, stains and mounts tissue specimens; and performs related work as required. Applicants must have at least 6 months experience in a histology lab. For immediate consideration, please email resume to: heidi.hawtho...@onassignment.commailto:heidi.hawtho...@onassignment.com. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Lab Assistants Embedding
I attended a seminar recently that was given by an Pathologist who was an experienced expert witness. The substance of the seminar addressed the top items that would be looked at when a pathology lab is involved in a lawsuit. One item that was specifically mentioned was that tissue orientation within the block is often examined as a potential reason for false negative surgical cases. The statistics mentioned were all for derm specimens and how when the block was sectioned through, it was reasoned that tissue orientation within the block was at fault. So, the sound advice given by all of you to document and train properly is vital. Steve -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Angela Bitting Sent: Friday, January 21, 2011 9:01 AM To: histonet@lists.utsouthwestern.edu; histonet-reque...@lists.utsouthwestern.edu Subject: [Histonet] Lab Assistants Embedding Hello Ever-helpful Histofriends, I want to train my lab assistants to embed simple tissues like breast resection specimens, placentas, etc. My manager feels that this will break some kind of regulations and won't sit well with our doctoral staff. I feel pretty confident that other Labs are doing this and I'm ready to take up the torch, but I need some data from other hospitals. Are any of you utilizing your non-HT staff to do these tasks and what hoops did you have to jump through to get approval from the Doctoral staff? In addition, how does CAP look at this? Thanks for your help, as always, Angie Angela Bitting, HT(ASCP), QIHC Technical Specialist, Histology Geisinger Medical Center 100 N Academy Ave. MC 23-00 Danville, PA 17822 phone 570-214-9634 fax 570-271-5916 No trees were hurt in the sending of this email However many electrons were severly inconvienienced! IMPORTANT WARNING: The information in this message (and the documents attached to it, if any) is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, distribution or any action taken, or omitted to be taken, in reliance on it is prohibited and may be unlawful. If you have received this message in error, please delete all electronic copies of this message (and the documents attached to it, if any), destroy any hard copies you may have created and notify me immediately by replying to this email. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] fibroblast marker in mouse tissue
Hi All, What is everyone using to stain for fibroblasts in frozen mouse tissue? Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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] Mohs
I'm in need of some info from Mohs Techs. Average time to cut 1 patient? How many patients a day? Anybody cut or get multiple patients at the same time? I would love to hear from you ! Thank You ,Dawn Dawn Oakes HT Olympic Medical Center - Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended individual(s) named above and may contain confidential, privileged, and/or protected information. Any unauthorized review, use, disclosure, copying, or distribution of its contents is prohibited. If you are not the intended recipient, you have received this email in error. If so, please notify the sender immediately by reply email and delete/destroy the original and all copies of this communication. Also know that Internet e-mail is not secure. In choosing to communicate with Olympic Medical Center by email you will assume these confidentiality risks. Internet messages may become corrupted, incomplete, or may incorrectly identify the sender. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Banana smelling chemical in our formalin
Histonet, We get a number of our specimens from outreach clients. We received back from one of our sites a jug of Formalin to be recycled. However when it was opened up it had a strong odor of bananas. I know we shouldn't smell the chemicals, but luckily this was caught before it was put through our recycler. I was wondering if anyone might have an idea what this could be? Is there a manufacturer that adds an odor (like bananas) to their formalin? Is it a strange solvent? The idea of it being amyl acetate has been bounced around, but I cannot see a histologic use for such a chemical. Thanks for your input. -- Patrick Laurie HT(ASCP)QIHC CellNetix Pathology Laboratories 1124 Columbia Street, Suite 200 Seattle, WA 98104 plau...@cellnetix.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Re: Banana smelling chemical in our formalin
That wasn't formalin. It was Histologie for Men, the new banana-scented cologne for histotechs. But seriously, I guess it's formalin ethyl acetate, which smells vaguely banana-like and is used for extraction of parasites from stool samples. Adam On Mon, Jan 24, 2011 at 4:56 PM, Patrick Laurie foreig...@gmail.com wrote: Histonet, We get a number of our specimens from outreach clients. We received back from one of our sites a jug of Formalin to be recycled. However when it was opened up it had a strong odor of bananas. I know we shouldn't smell the chemicals, but luckily this was caught before it was put through our recycler. I was wondering if anyone might have an idea what this could be? Is there a manufacturer that adds an odor (like bananas) to their formalin? Is it a strange solvent? The idea of it being amyl acetate has been bounced around, but I cannot see a histologic use for such a chemical. Thanks for your input. -- Patrick Laurie HT(ASCP)QIHC CellNetix Pathology Laboratories 1124 Columbia Street, Suite 200 Seattle, WA 98104 plau...@cellnetix.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] gram stain
Does anyone have a method for the Twort stain or a gram stain that does not use picric acid or ether? We were using a Twort stain and I cannot find the original reference for it and we suspect we have one of the concentrations wrong. Thank you, Jennifer MacDonald ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Archives
Hi - I go by subject matter, but there was a time (a very long time ago) where they had them by date as well. When in doubt I just wing it and somehow I find what I'm looking for. Vikki On Jan 24, 2011 7:14 PM, Joe Nocito jnoc...@satx.rr.com wrote: Greetings all, I'm going to show my ignorance (like that hasn't happened before). When accessing the archives, how do I do it? Do I search by topic or date? All the time I've been on here, I never had to use the archives. Thanks. Oh Oh, I feel another tear coming Joe ___ 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] gram stain
Thank you!! Tony Henwood antho...@chw.edu.au Sent by: histonet-boun...@lists.utsouthwestern.edu 01/24/2011 04:54 PM To 'Jennifer MacDonald' jmacdon...@mtsac.edu, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu cc Subject RE: [Histonet] gram stain Jennifer, Here is the method from our manual: Gram Stain Principle: Crystal violet stains up both Gram positive and Gram negative bacterial wall but only the Gram positive wall structure is capable of retaining the violet molecules that are locked in by iodine. Gram negative cell wall loses the violet colour when differentiated with acetone, and stains up red as a result of counterstaining. Fixation: 10% buffered formalin. Microtomy: Paraffin sections at 5µm. Controls: Placenta containing gram positive cocci and gram negative bacilli Reagents: 1. 1% Crystal Violet - Warning: Flammable, Suspected Carcinogen - see MSDS Crystal Violet (Or Methyl Violet) 10g Ethanol 100 ml 1% Ammonium Oxalate 400 ml 2. Lugol's Iodine - Warning: Toxic - see MSDS 3. Acetone - Warning: Flammable - see MSDS 4. Twort's counterstain: 0.2% neutral red (CI 50040) in ethanol 9ml 0.2% fast green (CI 42053) in ethanol1ml Distilled water 30ml Mix immediately before use. Procedure: 1. Bring sections to distilled water. 2. Cover sections with crystal violet 30-60 seconds 3. Rinse slides in water 4. Stain with Lugol's iodine 30-60 seconds 5. Tap water wash thoroughly 6. Blot sections lightly to remove excess water 7. Differentiate with acetone until dye stops running off the section. 8. Wash thoroughly with water 9. Counterstain Tworts 10 minutes 10. Wash, dehydrate quickly, clear and mount. Results: Gram positive bacteria violet blue Gram negative bacteria red Surrounding connective tissuegreen Reference: 1. Preston, Morrell. J. Path. Bact. (1962), 84:241-5. 2. Twort, J. State. Medicine (1924), 32:351. 3. Cherukian, Schenk. J. Histotech. (1982), 5(3):127-128. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 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 Jennifer MacDonald Sent: Tuesday, 25 January 2011 9:15 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] gram stain Does anyone have a method for the Twort stain or a gram stain that does not use picric acid or ether? We were using a Twort stain and I cannot find the original reference for it and we suspect we have one of the concentrations wrong. Thank you, Jennifer MacDonald ___ 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 mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] in situ - thanks 1 more query
Hi all, just wanted to say thanks for all the info received. L Looking through the protocols it appears that i need an anti-DIG antibody, preferably HRP labelled - any suggestions as to supplier? I remember using one from DAKO, but don't see it in the latest catalogue. Please - any sugestions as to where I can get it? Thanks again - you guys (and gals) rock!! -- Louise Renton Bone Research Unit University of the Witwatersrand Johannesburg South Africa +27 11 717 2298 (tel fax) 073 5574456 (emergencies only) There are nights when the wolves are silent and only the moon howls. George Carlin No trees were killed in the sending of this message. However, many electrons were terribly inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
