[Histonet] RE: post FACS staining
Melissa Mazan
melissa.mazan <@t> tufts.edu
Fri Aug 10 07:26:35 CDT 2007
Hi all,
Thanks so much for your advice. On your questions:
We sort a whole lung cell suspension based on CD45, CD31 negative, sca-1
positive. I have hoped that as these are cell surface markers and I"m
staining for cytoplasmic antigens, I would be able to distinguish
between leftover Ab (which should only be the sca-pos, as the others are
a negative sorting strategy) and the proteins I"m looking for. If I look
at the cytos before any further staining, I often see a fluorescent rim
around the cell, which is what sca-1 should look like. The antibodies
that I am using work very well on mouse tissues and on Western blot. For
pro SP-C, in particular, my positive control (ATII cells) fluoresce very
brightly and are reminiscent of tissue cells - punctate color throughout
the cytoplasm. What is confusing is that my negative control (serum
only) does not fluoresce beyond the faint rim of stain from leftover
sca-1 staining, (all well and good) but my C45neg/CD31neg/Sca-1 pos
cells have a weak fluorescence that is distinctly different to neg
control, but nowhere near pos control. When I stain with CC10, I have a
similar result, although I do not have a good pos control other than
tissue (no post FACS pos control). It would be nice if this were real,
but when I stain the CD45neg/CD31neg/Sca-1neg fraction, I get the same
result -which makes no sense. I wonder if the FACS is changing, for
instance, the charge on the cells sufficiently, or is perhaps changing
the permeability of the cell sufficiently to cause this sort of
non-specific staining?
I have tried methanol/acetic acid - didn't like what it did to the
morphology of the cells; acetone alone; acetone and cytofix/cytoperm,
and cytofix/cytoperm alone.
I agree with the increased background with the BSA - however, its
difficult to keep good morphology of the cells without.
I'm coming to the conclusion that post-FACS immuncytochemistry may be a
bad idea...Melissa
histonet-request <@t> lists.utsouthwestern.edu wrote:
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> Today's Topics:
>
> 1. Histonet Digest, Vol 45, Issue 12 (ryan <@t> upei.ca)
> 2. RE: immunostaining post-FACS (Tarango, Mark)
> 3. Re: Masson's Trichrome Troubleshooting (John Kiernan)
> 4. Re: Golgi stainembedding (John Kiernan)
> 5. change in email address (Dawn Cowie)
> 6. Re: change in email address (LINDA MARGRAF)
> 7. RE: NSH catalog (Bartlett, Jeanine (CDC/CCID/NCZVED))
> 8. NSH Symposium/Convention (Aubrey Wanner)
> 9. Re: change in email address (Joe Nocito)
> 10. Slide Staining Variability (Annette Hall)
> 11. (no subject) (Bruijntjes, J.P. (Joost))
> 12. Slide Staining Variability (Judy Collins)
> 13. Ethanol, never methanol for CD marker fixation RE: [Histonet]
> immunostaining post-FACS (Gayle Callis)
> 14. RE: Slide Staining Variability (Mike Pence)
> 15. Region II Fall Seminar for September 6, 7 and 8, 2207
> (Pamela Marcum)
> 16. Masson's Troubleshooting (Ford, Judi)
> 17. Job Posting - Research Histology, Kansas City, MO (Johnson, Teri)
> 18. histology contractor needed (Human Resources)
> 19. NSH (Renko, Heather D.)
> 20. Blocking arrangements in research for different species
> (Wilson, Carol)
> 21. Re: Slide Staining Variability (Joe Nocito)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 8 Aug 2007 20:33:05 -0300
> From: <ryan <@t> upei.ca>
> Subject: [Histonet] Histonet Digest, Vol 45, Issue 12
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <8C0B2B7720B <@t> acad1.cs.upei.ca>
>
> C.L. Ryan will be out of the office from Friday Aug 10 until Monday Aug. 13, incl.
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 8 Aug 2007 17:08:08 -0700
> From: "Tarango, Mark" <mtarango <@t> nvcancer.org>
> Subject: RE: [Histonet] immunostaining post-FACS
> To: "Melissa Mazan" <melissa.mazan <@t> tufts.edu>,
> histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <5AEC610C1CE02945BD63A395BA763EDE011B6DB8 <@t> NVCIEXCH02.NVCI.org>
> Content-Type: text/plain; charset=us-ascii
>
> These cells haven't already been stained and sorted or something, have
> they? If so, I would assume that whatever antibodies they used for
> flow/FACS will be on the cells. You said you use cytofix/cytoperm, that
> might denature the flow antibodies (if there are any), but you never
> know.
>
> Have you tried doing the cytofix/cytoperm on the cells while they are in
> suspension, and THEN making the cytospins? Switching to a different
> fixative? Gayle Callis had a post a while ago about using a fixative of
> 75% acetone and 25% methanol for murine CD markers. Maybe it would work
> for all your markers. I've tried modifying this fixative using propanol
> instead of ethanol for frozen sections on human tissue. It worked.
>
> When all else fails, I like to, as closely as possible, treat the
> specimen like formalin-fixed paraffin-embedded tissue. I will fix in
> formalin, dehydrate in staining dishes (like processing tissue) ...then
> bring it back to water and do antigen retrieval before staining. You
> might lose too many cells trying this on cytospins though, but might as
> well throw it out there.
>
>
> Just some thoughts...
>
>
> Mark Adam Tarango HT(ASCP)
>
> Histology/Immunohistochemistry Supervisor
>
> Nevada Cancer Institute
>
> One Breakthrough Way
>
> Las Vegas, NV 89135
>
> mtarango <@t> nvcancer.org
>
> Direct Line (702) 822-5112
>
> Fax (702) 939-7663
>
>
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melissa
> Mazan
> Sent: Wednesday, August 08, 2007 4:16 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] immunostaining post-FACS
>
> Hi all, Wondering if anyone has done much immunostaining post FACS. WE
> have had a lot of trouble with this procedure - we collect our cells
> (from murine lung) into BSA on ice, bring them back to our lab (FACS
> is at our core facility, so it means about an hour and a half before
> the cells get to our lab). In the lab, we immediately cytospin - the
> morphology is nice on H and E - and then fix in Cytofix Cytoperm for 20
> minutes at 4C. We either commence immunostaining immediately after, or
> leave in fridge for the next day. We do either immunofluorescence or
> immunoenzyme (usually ABC system) for identifying antigens of interest.
> We always have a negative control, but don't always have a positive
> control. We do either double staining for SPC and CC10 or single
> staining for alpha sma, vimentin, e-cad, or pancytokeratin. The
> problem that we have is that although the negative controls are very
> negative with respect to the cases, we seem to be getting a lot of
> false positives when the antibody is actually applied - for instance,
> my cells will stain 80% positive - strongly positive - using alpha sma,
> but a Western of the same cells will show no alpha-sma at all, whereas
> there is positive staining on whole lung suspensions. Any ideas?
> Advice for getting good post-FACS immunostaining? Many thanks - Melissa
>
> Melissa R. Mazan, DVM, Diplomate ACVIM
> Associate Professor and Director of Equine Sports Medicine
> Department of Clinical Sciences
> Tufts Cumming School of Veterinary Medicine
> 200 Westborough Road
> North Grafton, MA 01536
> Tel:508-839-5395
> Fax:508-839-7922
> email: melissa.mazan <@t> tufts.edu
>
>
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>
> "EMF <nvcancer.org>" made the following annotations.
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> ------------------------------
>
> Message: 3
> Date: Thu, 09 Aug 2007 00:01:04 -0400
> From: John Kiernan <jkiernan <@t> uwo.ca>
> Subject: Re: [Histonet] Masson's Trichrome Troubleshooting
> To: "Ford, Judi" <judi.ford <@t> roche.com>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <ddfab957cd89.46ba5940 <@t> uwo.ca>
> Content-Type: text/plain; charset=us-ascii
>
> If the known positive control slides were side-by side with the study slides (which stained correctly), then there must be something wrong with the control slides, not the solutions or the steps of the method. Different fixation or inadequate removal of paraffin come to mind, but you've probably thought of those. Your study slides of heart etc were well stained, so perhaps this will be a suitable positive control for future Masson stainings.
>
> It would be satisfying, however, to know why previously positive collagen in your sections of muscle etc failed to stain with aniline blue. Was the collagen in these sections red or not stained at all? If it was red, then the PTA/PMA had failed to displace the red dye, and a longer time in PTA/PMA is likely to be needed. If that's the case the control sections are unsuitable for staining beside your study sections. The final wash is in dilute acetic acid to prevent the removal of any bound red or blue dye, which can occur if plain water is used. You rightly examined the wet sections at this stage, an action that shows that you know what you are about, unlike many professors, postdocs, graduate students, . . .
>
> Please let us all know if you find out why the collagen in previously good positive-control tissue became unstainable with the Masson's method in Luna's AFIP manual.
>
> John A. Kiernan
> Department of Anatomy & Cell Biology
> The University of Western Ontario
> London, Canada N6A 5C1
> http://publish.uwo.ca/~jkiernan/index.htm
> ---
> ----- Original Message -----
> From: "Ford, Judi" <judi.ford <@t> roche.com>
> Date: Wednesday, August 8, 2007 15:48
> Subject: [Histonet] Masson's Trichrome Troubleshooting
> To: histonet <@t> lists.utsouthwestern.edu
>
>
>> Hi everyone,
>>
>>
>>
>> I'm hoping someone can give me suggestions on what happened to
>> my stain.
>> I did a Masson's Trichrome on mouse hearts/aortas this
>> morning. I
>> followed the AFIP protocol exactly and used two different
>> control slides
>> (tongue, aorta, skeletal muscle). We have previously stained control
>> slides with the tissue I used this time and they worked beautifully.
>>
>>
>>
>> After finishing the glacial acetic acid rinse I examined the slides
>> under the scope and found that neither of the control slides stained
>> with the aniline blue, but all the study slides stained
>> beautifully with
>> aniline blue. I tried going back and restaining (from
>> phosphomolybdic/phosphotungstic acid) the slides and the same results
>> showed. I thought maybe I had them in the glacial acetic acid
>> too long
>> so I cut that back to 3 minutes instead of 5 for the second round.
>>
>>
>>
>> Any ideas on what could have happened? Could the control slides have
>> been sitting too long as unstained slides? Could the aniline blue
>> working solution need an extra punch from glacial acetic acid?
>> In the
>> past I've reused aniline blue without any problems and this solution
>> wasn't past its expiration date.
>>
>>
>>
>> Would love to hear your thoughts......
>>
>>
>>
>> Judi Ford
>>
>> Palo Alto, CA
>>
>>
>>
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>
>
> ------------------------------
>
> Message: 4
> Date: Thu, 09 Aug 2007 01:00:14 -0400
> From: John Kiernan <jkiernan <@t> uwo.ca>
> Subject: Re: [Histonet] Golgi stainembedding
> To: Bob Nienhuis <bob.nienhuis <@t> gmail.com>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <ddeac6d1e178.46ba671e <@t> uwo.ca>
> Content-Type: text/plain; charset=us-ascii
>
> Dear Bob N.
>
> If your archived tissue is in formaldehyde you probably will not be able to get Golgi preparations that are good enough for research purposes.
>
> There are plenty of published Golgi techniques for ideally fixed material (up to a few weeks).
>
> Researchers demanding perfect dendritic morphology (for counting dendritic spines, or making mathematical models of branching) often fix their specimens in Cox's solution and apply the alkaline developer to thick sections cut with a vibrating microtome (Vibratome or similar). This can provide superb Golgi preparations, even with the brains of adult animals. Kolb, at Lethbridge University, is a major proponent of these methods. His publications should be available in your library. Try Scopus or Web of Science.
>
> For good Golgi preparations you need to work closely with someone experienced with the various techniques and you must become familiar with the technical literature. Two books have chapters that serve as introductory reading:
> Bradley, PB (ed) 1975. Methods in Brain Research . Wiley.
> Santini, M. 1975. Golgi Centennial Symposium. Raven Press.
>
> There are two families of Golgi methods. The intracellular chromate ions may be precipitated by silver or mercury, and the results are not the same. I wish you well with obtaining good Golgi preps on fixed human brains. If you perfect the technique you will have the makings of a significant publication.
>
> John Kiernan
> Anatomy & Cell Biology
> University of Western Ontario
> London, Canada.
> ---
> ----- Original Message -----
> From: Bob Nienhuis <bob.nienhuis <@t> gmail.com>
> Date: Wednesday, August 8, 2007 14:59
> Subject: [Histonet] Golgi stainembedding
> To: histonet <@t> lists.utsouthwestern.edu
>
>
>> Anybody have suggestions for alternatives to celloidin embedding
>> for Golgi-Kopsch? Has anyone done it, and can point out some
>> pitfalls I might avoid in doing it? I want to Golgi stain some
>> archival human brain tissue.
>>
>> I have not tried it yet, and understand it can be a bit tricky.
>>
>> Bob
>> nienhuis <@t> ucla.edu
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 9 Aug 2007 05:35:20 -0700 (PDT)
> From: Dawn Cowie <dlcowie <@t> prodigy.net>
> Subject: [Histonet] change in email address
> To: histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <771388.95993.qm <@t> web81007.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
>
> Dear Histonet,
> please change my email address for receiving messages to Dawn_Cowie <@t> yahoo.com
>
> thanks,
> Dawn
>
>
> ------------------------------
>
> Message: 6
> Date: Thu, 09 Aug 2007 08:02:05 -0500
> From: "LINDA MARGRAF" <LINDA.MARGRAF <@t> childrens.com>
> Subject: Re: [Histonet] change in email address
> To: "histonet" <histonet <@t> lists.utsouthwestern.edu>, "Dawn Cowie"
> <dlcowie <@t> prodigy.net>
> Message-ID: <46BAC9FD020000DA000113A0 <@t> CNET3.CHILDRENS.COM>
> Content-Type: text/plain; charset=US-ASCII
>
> Dear Dawn (and other Histonetters):
> I will change your address on the Histonet email address list as you requested but I wanted to point out to you and all the other Histonet members that you can change your own address or change other features of your subscription by going to the website http://lists.utsouthwestern.edu/mailman/listinfo/histonet and using the instructions at the bottom of the page. People can switch to digest mode, temporarily stop messages while they go on vacation etc. If you cannot remember your password you can request it there too. For anyone having trouble posting messages because the server says you are not a member, this likely means your address has changed somewhat from when you subscribed (a common occurrence in institutional email systems) and you need to update your address which you can do there.
> By the way Histonet is up tp >2600 members now!
> Thanks,
> Linda M
> Histonet administrator
>
>
>>>> Dawn Cowie <dlcowie <@t> prodigy.net> 08/09/07 7:35 AM >>>
>>>>
> Dear Histonet,
> please change my email address for receiving messages to Dawn_Cowie <@t> yahoo.com
>
> thanks,
> Dawn
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
> ------------------------------
>
> Message: 7
> Date: Thu, 9 Aug 2007 09:12:29 -0400
> From: "Bartlett, Jeanine (CDC/CCID/NCZVED)" <jqb7 <@t> CDC.GOV>
> Subject: RE: [Histonet] NSH catalog
> To: "Kim Tournear" <kimtournear <@t> yahoo.com>, "Histonet"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <34BB307EFC9A65429BBB49E330675F7202BDBEF3 <@t> LTA3VS003.ees.hhs.gov>
> Content-Type: text/plain; charset=us-ascii
>
> I received mine a few weeks ago.
>
>
> Jeanine Bartlett
> Infectious Disease Pathology Branch
> (404) 639-3590
> jeanine.bartlett <@t> cdc.hhs.gov
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kim
> Tournear
> Sent: Wednesday, August 08, 2007 7:13 PM
> To: Histonet
> Subject: [Histonet] NSH catalog
>
> Am I the only one that hasn't received the NSH catalog for the symposium
> in Denver, or do we have to get it on line now?
>
> Kim Tournear, HT (ASCP), QIHC ( ASCP)
> Specialists in Dermatology
> Histology/Mohs Supervisor
> Tucson, AZ
>
>
>
>
> ---------------------------------
> Moody friends. Drama queens. Your life? Nope! - their life, your story.
> Play Sims Stories at Yahoo! Games.
> _______________________________________________
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
>
> ------------------------------
>
> Message: 8
> Date: Thu, 9 Aug 2007 09:26:15 -0400
> From: "Aubrey Wanner" <aubrey <@t> nsh.org>
> Subject: [Histonet] NSH Symposium/Convention
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <AE7E94A55D4BAB42B9658B508E1C37A33B4AFE <@t> 2kserver.nsh.local>
> Content-Type: text/plain; charset="us-ascii"
>
> The 33rd Annual NSH Symposium/Convention will take place in Denver,
> October 26-31. Registration programs mailed in May - if you did not
> receive a copy please feel free to contact the NSH Office, 443-535-4060
> or visit the website and click on Meetings/Events >
> Symposium/Convention.
>
> All workshops are still available and we still have rooms in the
> headquarters hotel. Any questions please call our office - we hope to
> see you there!
>
>
>
> Mrs. Aubrey M.J. Wanner
> Meeting Manager, Annual Symposium/Convention
> Managing Editor, Journal of Histotechnology
>
>
> We've Moved!
> National Society for Histotechnology
> 10320 Little Patuxent Parkway | Suite 804
> Columbia, MD 21044
> P | 443.535.4060
> Direct | 443.535.4065
> F | 443-535-4055
> <http://www.nsh.org/conventions/index.html> http://www.nsh.org/
>
>
>
>
> ------------------------------
>
> Message: 9
> Date: Thu, 9 Aug 2007 08:40:05 -0500
> From: "Joe Nocito" <jnocito <@t> satx.rr.com>
> Subject: Re: [Histonet] change in email address
> To: "LINDA MARGRAF" <LINDA.MARGRAF <@t> childrens.com>, "histonet"
> <histonet <@t> lists.utsouthwestern.edu>, "Dawn Cowie"
> <dlcowie <@t> prodigy.net>
> Message-ID: <000b01c7da8a$ccd435f0$d49eae18 <@t> yourxhtr8hvc4p>
> Content-Type: text/plain; format=flowed; charset="iso-8859-1";
> reply-type=original
>
> WOW! I think it's time again to thank Dr. M and her staff for maintaining
> the Histonet. Party in Denver!?
>
> ----- Original Message -----
> From: "LINDA MARGRAF" <LINDA.MARGRAF <@t> childrens.com>
> To: "histonet" <histonet <@t> lists.utsouthwestern.edu>; "Dawn Cowie"
> <dlcowie <@t> prodigy.net>
> Sent: Thursday, August 09, 2007 8:02 AM
> Subject: Re: [Histonet] change in email address
>
>
> Dear Dawn (and other Histonetters):
> I will change your address on the Histonet email address list as you
> requested but I wanted to point out to you and all the other Histonet
> members that you can change your own address or change other features of
> your subscription by going to the website
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet and using the
> instructions at the bottom of the page. People can switch to digest mode,
> temporarily stop messages while they go on vacation etc. If you cannot
> remember your password you can request it there too. For anyone having
> trouble posting messages because the server says you are not a member, this
> likely means your address has changed somewhat from when you subscribed (a
> common occurrence in institutional email systems) and you need to update
> your address which you can do there.
> By the way Histonet is up tp >2600 members now!
> Thanks,
> Linda M
> Histonet administrator
>
>
>>>> Dawn Cowie <dlcowie <@t> prodigy.net> 08/09/07 7:35 AM >>>
>>>>
> Dear Histonet,
> please change my email address for receiving messages to
> Dawn_Cowie <@t> yahoo.com
>
> thanks,
> Dawn
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
> ------------------------------
>
> Message: 10
> Date: Thu, 9 Aug 2007 08:41:50 -0500
> From: Annette Hall <Annette_hall <@t> pa-ucl.com>
> Subject: [Histonet] Slide Staining Variability
> To: Histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <9FC023A4AB52BB4D87DC6456081A822C012B6054 <@t> mercury.pa-ucl.com>
> Content-Type: text/plain
>
> Histonetters,
>
> For those of you who use automated IHC staining systems, has anyone had
> issues with part of the slide staining and the rest of the slide not? We
> have adopted the policy of placing the control on with the patient tissue as
> a more comprehensive check of the system. However, we occasionally find the
> control is acceptable but the patient fails to even counterstain. This has
> been an intermittent problem on the Ventana Benchmark for some time, but the
> frequency has been increasing to almost daily.
>
> Any thoughts?
>
> Thanks,
> Annette J Hall
> Histo/Cyto/Micro Supervisor
> United Clinical Laboratories
> Dubuque, IA 52001
>
> -----Original Message-----
> From: Harrison, Sandra C. [mailto:Sandra.Harrison3 <@t> va.gov]
> Sent: Monday, August 06, 2007 3:35 PM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Leica autostainer w/integrated coverslipper
>
> Histonetters,
> Could anyone tell me if they're using the Leica AutoStainer XL (ST5010)
> with the integrated coverslipper (CV5030)?
> Also, can you tell me if you've experienced any trouble with it and how
> long you've been using it?
>
> Thanks,
> Sandy
> _______________________________________________
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
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>
>
>
>
> ------------------------------
>
> Message: 11
> Date: Thu, 9 Aug 2007 16:34:53 +0200
> From: "Bruijntjes, J.P. (Joost)" <joost.bruijntjes <@t> tno.nl>
> Subject: [Histonet] (no subject)
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <8865601DD17A554CB489C17FFD8A51B248507E <@t> MAIL04.tsn.tno.nl>
> Content-Type: text/plain; charset="us-ascii"
>
> Please, change my email address into joost.bruijntjes <@t> tno.nl
>
>
>
> Thanks
>
>
>
> TNO.NL <http://www.tno.nl/>
>
> Joost Bruijntjes
>
> T +31 30 694 44 80
> F +31 30 694 49 86
> E joost.bruijntjes <@t> tno.nl <mailto:joost.bruijntjes <@t> tno.nl>
>
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>
>
> This e-mail and its contents are subject to the DISCLAIMER at http://www.tno.nl/disclaimer/email.html
>
> ------------------------------
>
> Message: 12
> Date: Thu, 9 Aug 2007 10:35:05 -0400
> From: "Judy Collins" <JCollins <@t> palmbeachpath.com>
> Subject: [Histonet] Slide Staining Variability
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <F8D33B6C3F7BA143998A51C5CF6EE09F2E95B5 <@t> pbpsflexch01.pbp.local>
> Content-Type: text/plain; charset="us-ascii"
>
> With the Ventana system, staining variability on different parts of the
> slide usually is a result of a problem with the vortex mixers, although
> other issues such as dispensers not working properly can cause this as
> well. You can run a test on the vortex mixers (Find the procedure in
> the online manual). You can also call their technical support line for
> assistance.
>
> Judy Collins
>
>
>
> ------------------------------
>
> Message: 13
> Date: Thu, 09 Aug 2007 08:50:49 -0600
> From: Gayle Callis <gcallis <@t> montana.edu>
> Subject: Ethanol, never methanol for CD marker fixation RE: [Histonet]
> immunostaining post-FACS
> To: "Tarango, Mark" <mtarango <@t> nvcancer.org>,
> Histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <6.0.0.22.1.20070809082551.01b37dd8 <@t> gemini.msu.montana.edu>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
>
> Dear Mark and Melissa,'
>
> A correction to Marks reply. I do NOT use methanol in my acetone/alcohol
> fixative. The fixative is made up with absolute ethanol 75% acetone/25%
> absolute ETHANOL. Methanol ruins my CD markers (in the literature and also
> in Histonet archives) . However, this acetone alcohol fixative is not good
> with human CD4 and CD8 markers since they do not like ethanol either. And
> there are antibodies that will work best with formalin or paraformaldehyde
> fixation.
>
> Could it be the antigens you are trying to stain do not like the fixative
> you are using? I suggest you do a fixation panel on known positive cells
> to optimize the fixation. That positive control is important to know IF
> your antibodies, and method is working. A known positive tissue control
> may help too, then you know your antibodies are working on mouse tissues,
> cells. Frozen sections and different fixatives should provide some
> answers. Some antibodies work better on Western blots, but when you try to
> use them on tissue sections (or at the same concentration as the blot) you
> get nothing. Also, we try to purchase antibodies known to work on
> cells/tissue sections, and avoid just the Western Blot application.
>
> Double check your BSA, and make sure it is protease/immunoglobulin free,
> Jackson has this - there is an interesting publication on albumin causing
> background staining in AIMM journal, titled Albumin in
> Immuhohistochemistry: Foe or Friend? 14(4l ), December 2006 Mittelbronn M
> et al. I do have the publication in pdf form if you would like to read it.
>
> I am curious and as Mark pointed out - to sort the cells, are you staining
> those with an antibody, sort, collect, then stain with another
> antibody? I was a bit lost on this.
>
> Gayle Callis
> MT,HT,HTL(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
>
>
>
>
>
> At 06:08 PM 8/8/2007, you wrote:
>
>> These cells haven't already been stained and sorted or something, have
>> they? If so, I would assume that whatever antibodies they used for
>> flow/FACS will be on the cells. You said you use cytofix/cytoperm, that
>> might denature the flow antibodies (if there are any), but you never
>> know.
>>
>> Have you tried doing the cytofix/cytoperm on the cells while they are in
>> suspension, and THEN making the cytospins? Switching to a different
>> fixative? Gayle Callis had a post a while ago about using a fixative of
>> 75% acetone and 25% methanol for murine CD markers. Maybe it would work
>> for all your markers. I've tried modifying this fixative using propanol
>> instead of ethanol for frozen sections on human tissue. It worked.
>>
>> When all else fails, I like to, as closely as possible, treat the
>> specimen like formalin-fixed paraffin-embedded tissue. I will fix in
>> formalin, dehydrate in staining dishes (like processing tissue) ...then
>> bring it back to water and do antigen retrieval before staining. You
>> might lose too many cells trying this on cytospins though, but might as
>> well throw it out there.
>>
>>
>> Just some thoughts...
>>
>>
>
>
>
>
>
>> Mark Adam Tarango HT(ASCP)
>>
>> Histology/Immunohistochemistry Supervisor
>>
>> Nevada Cancer Institute
>>
>> One Breakthrough Way
>>
>> Las Vegas, NV 89135
>>
>> mtarango <@t> nvcancer.org
>>
>> Direct Line (702) 822-5112
>>
>> Fax (702) 939-7663
>>
>>
>>
>>
>> -----Original Message-----
>> From: histonet-bounces <@t> lists.utsouthwestern.edu
>> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melissa
>> Mazan
>> Sent: Wednesday, August 08, 2007 4:16 PM
>> To: histonet <@t> lists.utsouthwestern.edu
>> Subject: [Histonet] immunostaining post-FACS
>>
>> Hi all, Wondering if anyone has done much immunostaining post FACS. WE
>> have had a lot of trouble with this procedure - we collect our cells
>> (from murine lung) into BSA on ice, bring them back to our lab (FACS
>> is at our core facility, so it means about an hour and a half before
>> the cells get to our lab). In the lab, we immediately cytospin - the
>> morphology is nice on H and E - and then fix in Cytofix Cytoperm for 20
>> minutes at 4C. We either commence immunostaining immediately after, or
>> leave in fridge for the next day. We do either immunofluorescence or
>> immunoenzyme (usually ABC system) for identifying antigens of interest.
>> We always have a negative control, but don't always have a positive
>> control. We do either double staining for SPC and CC10 or single
>> staining for alpha sma, vimentin, e-cad, or pancytokeratin. The
>> problem that we have is that although the negative controls are very
>> negative with respect to the cases, we seem to be getting a lot of
>> false positives when the antibody is actually applied - for instance,
>> my cells will stain 80% positive - strongly positive - using alpha sma,
>> but a Western of the same cells will show no alpha-sma at all, whereas
>> there is positive staining on whole lung suspensions. Any ideas?
>> Advice for getting good post-FACS immunostaining? Many thanks - Melissa
>>
>> Melissa R. Mazan, DVM, Diplomate ACVIM
>> Associate Professor and Director of Equine Sports Medicine
>> Department of Clinical Sciences
>> Tufts Cumming School of Veterinary Medicine
>> 200 Westborough Road
>> North Grafton, MA 01536
>> Tel:508-839-5395
>> Fax:508-839-7922
>> email: melissa.mazan <@t> tufts.edu
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>> "EMF <nvcancer.org>" made the following annotations.
>> ------------------------------------------------------------------------------
>> CONFIDENTIALITY NOTICE: This e-mail 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 e-mail message or its attachments. If you
>> believe you have received this e-mail message in error,
>> please contact the sender by reply e-mail and destroy all
>> copies of the original message
>> ==============================================================================
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
> Gayle Callis
> MT,HT,HTL(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
>
>
>
>
>
>
> ------------------------------
>
> Message: 14
> Date: Thu, 9 Aug 2007 09:59:47 -0500
> From: "Mike Pence" <mpence <@t> grhs.net>
> Subject: RE: [Histonet] Slide Staining Variability
> To: "Annette Hall" <Annette_hall <@t> pa-ucl.com>,
> <Histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <661949901A768E4F9CC16D8AF8F2838CA1C6AE <@t> IS-E2K3.grhs.net>
> Content-Type: text/plain; charset="us-ascii"
>
> We have had some of these same problems and it is always been a nozzle
> or mixer problem. Do you perform regular PM's? The reaction buffer ph
> could also be off, that can cause varied staining.
>
> Mike
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Annette
> Hall
> Sent: Thursday, August 09, 2007 8:42 AM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Slide Staining Variability
>
>
> Histonetters,
>
> For those of you who use automated IHC staining systems, has anyone had
> issues with part of the slide staining and the rest of the slide not? We
> have adopted the policy of placing the control on with the patient
> tissue as a more comprehensive check of the system. However, we
> occasionally find the control is acceptable but the patient fails to
> even counterstain. This has been an intermittent problem on the Ventana
> Benchmark for some time, but the frequency has been increasing to almost
> daily.
>
> Any thoughts?
>
> Thanks,
> Annette J Hall
> Histo/Cyto/Micro Supervisor
> United Clinical Laboratories
> Dubuque, IA 52001
>
> -----Original Message-----
> From: Harrison, Sandra C. [mailto:Sandra.Harrison3 <@t> va.gov]
> Sent: Monday, August 06, 2007 3:35 PM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Leica autostainer w/integrated coverslipper
>
> Histonetters,
> Could anyone tell me if they're using the Leica AutoStainer XL (ST5010)
> with the integrated coverslipper (CV5030)? Also, can you tell me if
> you've experienced any trouble with it and how long you've been using
> it?
>
> Thanks,
> Sandy
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> NOTICE: This email may contain legally privileged information. The
> information is for the use of only the intended recipient(s) even if
> addressed incorrectly. If you are not the intended recipient, please
> notify the sender that you have received it in error and then delete it
> along with any attachments. Thank you.
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
>
> ------------------------------
>
> Message: 15
> Date: Thu, 09 Aug 2007 11:08:11 -0400
> From: Pamela Marcum <pmarcum <@t> vet.upenn.edu>
> Subject: [Histonet] Region II Fall Seminar for September 6, 7 and 8,
> 2207
> To: histonet <@t> lists.utsouthwestern.edu
> Cc: "Gloria Limetti":;
> Message-ID: <6.2.5.6.2.20070809110548.01c7c128 <@t> vet.upenn.edu>
> Content-Type: text/plain; charset="iso-8859-1"; format=flowed
>
>
>
> We are moving close to the dates for the meeting
> and registration is coming so please look over
> the program and request your full program if you
> don't have it. The following is a list of our
> topics and speakers. Please let us know if you need more information.
>
> The meeting is at the Delaware Technical
> Community College in Stanton DE. It is just off
> I-95 with easy access. The Thursday is primarily
> slated for management issues and topics while the
> remainder of Friday and Saturday are general
> topics for histology, cytology and research. If
> you CEUs and can't get to Denver this is an
> option for you to catch up this year.
>
> Region II Fall Symposium, September 6th, 7th, 8th 2007
>
> Delaware Technical Community College-Stanton, DE
>
> Can’t get to Denver for the NSH Symposium and Convention yet still need
> CEU’s? Join us for the Region II Fall Symposium in Delaware!
>
> Thursday Seminars*^ - will be geared toward management topics.
>
> 8:30 am to 11:30 am
> WS #01 CPT Coding-How Do I Code This
> One? Bonnie Whitaker 3 hrs
> WS #02 CAP Inspection-What’s New and Required Now Charlie Dorner 3 hrs
>
> 1:00 pm to 4:30 pm
> WS #03 Lean – Organizing Your Life and
> Lab Donna Montegue 3 hrs
>
> 1:00 pm to 2:30 pm
> WS #04 Reducing Immunohistochemistry
> Expenses Joe Myers 1.5 hrs
>
> 3:00 pm to 4:30 pm
> WS #05 Important Consideration in Selection
> of Joe
> Myers 1.5
> hrs IHC
> Instruments
>
>
> Friday Seminars*^
>
> 8:30 am to 12:00 pm
> WS #06 Beginning IHC (new to the field or
> refresher) Bonnie Whitaker 3 hrs
>
> 8:30 am to 10:00 am
> WS #07 Delaware Coroner
> Cases
> Dr. Richard Callery 1.5 hrs
> WS #08 Brain Diseases and Identification by
> Histology Dr. Barbara Crain 1.5 hrs
> WS #09 Sentinel Nodes & Breast
> Biopsies Dr. Mary Iococca 1.5 hrs
>
> 10:30 am to 12:00 pm
> WS #10 Tumor
> Classification
> Dr. Gary Witkin 1.0 hrs
> WS #11 Deconvolution
> Microscopy
> Dr. Robert Akins 1.5 hrs
> WS #12 Immunofluorescence for Kidney
> Biopsies Dr. Robert Garola 1.5 hrs
>
> 1:00 pm to 4:30 pm
> WS #13 Microwave
> Processing Connie Wavrin 3 hrs
> WS #14 Molecular
> Biology
> Abraham Joseph 3 hrs
>
> *Classes Subject to Change
> ^ CEU’s Pending Approval from NSH
>
>
>
>
> 1:00 pm to 2:30 pm
> WS #15 Round Table- to Discuss Options in
> Histology-Several Speakers will
> discuss other options for careers in
> histology, such as Pharmaceutical Industry,
> Veterinary & Research or Industry Options for
> Sales and Technical Training 1.5 hrs
> WS #16 ISH, FISH,
> PCR
> Mitch Gore 1.5 hrs
> WS #17 FNA
> Cytology
> Osman Ouattara 1.5 hrs
>
> 3:00 pm to 4:30 pm
> WS #18 Bog People of Northern
> Europe Sandra Olson PhD 1.5 hrs
>
> Saturday Seminars*^
>
> 8:30 am to 12:00 pm
> WS #19 IHC-Locating and Validating New Antibodies
> and IHC
> Reagents
> Charlie Dorner 3 hrs
> WS #20
> Ergonomics
> Jan Minchew 3 hrs
>
> 8:30 am to 10:00 am
> WS #21 Non-healing Lesion-Delusion or
> Parasite? David Brenner 1.5 hrs
> WS #22 Forensic & Medical
> Entomology Carolyn & Vincent
> D’Amico
> 1.5 hrs
>
> 10:30 am to 12:00 pm
> WS #23 Interesting Cases in Veterinary
> Pathology Dr. Perry Habecker 1.5 hrs
> WS #24 Clinical Flow
> Cytometry
> Brenda Rabeno 1.5 hrs
>
> 1:00 pm to 4:30 pm
> WS #25 IHC for Animal Histology (24
> limit-wet) Zahra Naser 3 hrs
> WS #26 Safety for
> Histology
> Sylvia Casey 3 hrs
> WS #27 Grossing for
> Histology Connie Wavrin 3 hrs
>
> 1:00 pm to 2:30 pm
> WS #28 Mad Cow Disease & Other
> Transmissible Dr. Michael Kanzer 1.0 hrs
> Spongiform Encephalopathies
> 3:00 pm to 4:30 pm
> WS #29 Lab Math and
> Chemistry
> Donna Montegue 1.5 hrs
>
> 8:30 am to 4:30 pm
> WS #30 QIHC
> Preparation
> Ethel Macrea 6 hrs
> WS #31 HT
> Readiness
> Linda Foster-Brown 6 hrs
>
>
> * Classes subject to change
> ^ CEU’s Pending Approval from NSH
>
> Fees
> Students & Retirees**
>
> ½ Day Session $
> 40.00 ½ Day Session $ 20.00
> Full Day Session $
> 80.00 Full Day Session $ 40.00
>
> Meet and Greet- seminar attendees included,
> guests $25.00
> Registration Fee $25.00 –waived if registered by August 15th
> 10% off groups of four or more from same laboratory in attendance
> All workshops are subject to change
> **proper student ID must be mailed with registration
> ________________________________________________________________________
>
> Full Program with abstracts will be available
> soon. If you would like to register now please fill in the following:
>
> Name ______________________________________
> Address ____________________________________
> ____________________________________
> ____________________________________
> Place of employment _________________________
> Home # ________________ Work# _____________
> E-mail _____________________________________
> ________________________________________________________________________
>
> Workshops planning to attend
>
> Thursday September 6th _________ _________ _________
> Friday September 7th _________ _________ _________ _________
> Saturday September 8th _________ _________ _________ _________
> Number planning to attend Meet and Greet ________
> Total amount enclosed ___________
> ________________________________________________________________________
>
> Please send completed forms with payment to:
> HSD
> PO Box 5341
> Wilmington, DE 19808-0341
>
> Any questions please contact: Michelle Hart at
> <mailto:mhart <@t> christianacare.org>mhart <@t> christianacare.org
> Elaine
> Perkins at <mailto:eperkins <@t> christianacare.org>eperkins <@t> christianacare.org
> Kristen
> Broomall at <mailto:histotechkb <@t> gmail.com>histotechkb <@t> gmail.com
> You may also contact your local histology
> societies for information concerning the 2007
> Region II Fall Symposium being held at DTCC in Stanton, Delaware.
>
> Hotels in the area of DTCC Stanton Campus
>
>
> Courtyard by Marriot-48 Geoffrey Dr.
> 302-456-3800
> · Block of rooms being held at
> $114.00/single and $124.00/double until August 9th
> Christiana Hilton-100 Continental Dr.
> 302-454-1500
> · Block of rooms being held at $129.00/single and $149.00/double until
> August 6th
> Days Inn-900 Churchman’s Rd.
> 302-368-2400
> Country Inn and Suites-1024 Old Churchman’s Rd.
> 302-266-6400
> Fairfield Inn by Marriot-65 Geoffrey Dr.
> 302-292-1500
> Red Roof Inns-415 Stanton-Christiana Rd.
> 302-292-2870
> Homestead Studio Suites Hotel-333 Continental Dr.
> 302-283-0800
>
>
>
> Best Regards,
>
> Pamela A Marcum
> Manager, Histology Special Procedures
> University of Pennsylvania
> School of Veterinary Medicine
> R.S. Reynolds Jr. CORL
> New Bolton Center
> 382 West Street Road
> Kennett Square, PA 19348
>
> Phone - 610-925-6278
> Fax - 610-925-8120
> E-mail - pmarcum <@t> vet.upenn.edu
>
>
>
>
> ------------------------------
>
> Message: 16
> Date: Thu, 9 Aug 2007 08:09:46 -0700
> From: "Ford, Judi" <judi.ford <@t> roche.com>
> Subject: [Histonet] Masson's Troubleshooting
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <DF7DFE066B39BB429DFBCCC82FFB7AB905D4C303 <@t> rplmsem1.nala.roche.com>
> Content-Type: text/plain; charset="us-ascii"
>
> Good morning to all from not so sunny northern CA:
>
>
>
> I want to thank you for all the input you gave me about my problem with
> the trichrome, especially to John, Gayle, Gundrun and Sarah. I
> re-examined the slides and compared the control with a Gomori's One-Step
> control slide, which had be stained a couple days prior. Both stains
> used the same control tissue. When I first looked at the Gomori's slide
> the collagen appeared to be stained well with the green. The same area
> in the Masson's didn't stain at all with the aniline blue. Upon
> examining both slides this morning I noticed that even though the green
> stain showed there was still some red stain within it. So, we are
> ordering new phosphotungstic acid and I'm cutting new slides. My
> co-worker is staining Masson's this morning with some other control
> slides and I'll try again with the new slides. If we're having the same
> results then I'm assuming that its got to be the phosphotungstic acid
> and we'll try again when the new stuff arrives.
>
>
>
> Anyway, I really appreciate all of your suggestion and input. This board
> has always been a valuable resource for me.
>
>
>
> Cheers!
>
> Judi Ford
>
> Palo Alto, CA
>
>
>
> ------------------------------
>
> Message: 17
> Date: Thu, 9 Aug 2007 10:22:58 -0500
> From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
> Subject: [Histonet] Job Posting - Research Histology, Kansas City, MO
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <C28BAF593DC3314E9C0F3A50191C2E7807A3E354 <@t> EXCHKC03.stowers-institute.org>
>
> Content-Type: text/plain; charset="us-ascii"
>
> The Stowers Institute for Medical Research has an opening for a
> Histology Specialist II to provide high quality research histology
> services.
>
> Responsibilities include sample preparation including sample
> receipt, fixation, processing, embedding, microtomy and staining;
> operation and maintenance of histology prep and ancillary equipment;
> training researchers in the use of common equipment and preparation
> techniques; and development of protocols for specialized research
> histology-related projects.
>
> In addition to the ability to effectively function in a
> team-oriented environment, the successful candidate must have fine-motor
> skills to manipulate tiny tissue samples and to demonstrate histology
> techniques; excellent communication skills to interact with the research
> scientists; a familiarity with basic biology, cell biology or genetics;
> and a record of excellent general laboratory skills.
>
> Minimum requirements include an Associates Degree in Biology or a
> related field and two years experience in basic research using histology
> techniques. Preference will be given to individuals with a Bachelors
> Degree in a related field, and or additional experience in basic
> research histology.
>
> For benefits information and to apply:
> http://www.stowers-institute.org/ScientistsSought/ScientistsSought.asp#r
> esume
>
> Teri Johnson, HT(ASCP)QIHC
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, MO 64110
>
>
>
>
> ------------------------------
>
> Message: 18
> Date: Thu, 9 Aug 2007 08:58:31 -0700
> From: "Human Resources" <histocontract <@t> gmail.com>
> Subject: [Histonet] histology contractor needed
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <dd0aacd00708090858o30dc479ay73fdc3e1d28e32e9 <@t> mail.gmail.com>
> Content-Type: text/plain; charset=WINDOWS-1252
>
> Temporary Histology Contractor needed – 4-6 months
>
>
>
> We are a well-known biotechnology company located in Silicon Valley, CA. We
> are looking for an experienced histotechnician to help out in our histology
> lab in a dynamic research setting. The successful candidate should have
> experience with basic histology procedures such as tissue trimming,
> processing, paraffin embedding, cutting, and staining with H&E, as well as
> experience with common special stains, and a facility with cryosectioning.
> Qualified candidates must have a minimum of 3-5 years relevant experience.
> Certification in HT and in phlebotomy would be a plus.
>
>
>
> To apply, please send resume to
> *histocontract <@t> gmail.com*<histocontract <@t> gmail.com>
> *,* along with desired hourly rate of pay.
>
>
> ------------------------------
>
> Message: 19
> Date: Thu, 9 Aug 2007 11:08:21 -0500
> From: "Renko, Heather D." <Heather.D.Renko <@t> osfhealthcare.org>
> Subject: [Histonet] NSH
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <40026EDDE64CDA47AB382C52619ACD3C0777558A <@t> pmc-rfd-mx01.intranet.osfnet.org>
>
> Content-Type: text/plain; charset=iso-8859-1
>
> I received my packet in June and registered. Check your membership expiration possibly?
>
> Heather Renko, Histology Coordinator
> OSF Saint Anthony Medical Center
> 5666 East State Street
> Rockford, Illinois 61108
> 815-395-5410
> Heather.D.Renko <@t> osfhealthcare.org
>
>
> ==============================================================================
> The information in this message is confidential and may be legally privileged. Access to this message by anyone other than the addressee is not authorized. If you are not the intended recipient, or an agent of the intended recipient, any disclosure, copying, or distribution of the message or any action or omission taken by you in reliance on it, is prohibited and may be unlawful. If you have received this message in error, please contact the sender immediately and permanently delete the original e-mail, attachment(s), and any copies.
> ==============================================================================
>
>
> ------------------------------
>
> Message: 20
> Date: Thu, 9 Aug 2007 12:23:24 -0400
> From: "Wilson, Carol" <wilson_c <@t> ricerca.com>
> Subject: [Histonet] Blocking arrangements in research for different
> species
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <9D443EB9D0270143B5AAF190CB1A58A305064EB2 <@t> dogwood.ricerca.com>
> Content-Type: text/plain; charset="us-ascii"
>
> I was wondering if anyone out there might be willing to share their
> blocking arrangement for different species in research with me via email
> or fax. Or a specific resource that I could find online, book, etc..
> I've googled without much success so far. I need somewhere to start and
> any and all help would be appreciated.
>
> Thanks,
>
> Carol
>
>
>
> Carol Wilson
>
> Team Leader - Histology
>
> Ricerca Biosciences, LLC
>
> 7528 Auburn Rd.
>
> Concord, OH 44077
>
> 440-357-3930
>
>
>
>
>
> ------------------------------
>
> Message: 21
> Date: Thu, 9 Aug 2007 11:51:02 -0500
> From: "Joe Nocito" <jnocito <@t> satx.rr.com>
> Subject: Re: [Histonet] Slide Staining Variability
> To: "Mike Pence" <mpence <@t> grhs.net>, "Annette Hall"
> <Annette_hall <@t> pa-ucl.com>, <Histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <003d01c7daa5$7a4f8800$d49eae18 <@t> yourxhtr8hvc4p>
> Content-Type: text/plain; format=flowed; charset="iso-8859-1";
> reply-type=original
>
> we have had the same problem on all three machines. To date, it has been:
> technician error, failure to remove a yellow locking ring, vortex mixer,
> slide warmer not hot enough, slide warmer too hot, dispenser not working,
> Venus is not aligned with Mars. Most of the time we have had to get a
> service rep out to repair something or other. One machine has had the
> carousel replaced twice. Need I say more?
> Oh yeah, I know you're here, go call my employer like you did last time.
> Oh, I should say "former" employer.
> Once again, the views of this editorial are my own and I have not been
> coerced, drugged, or beaten with a feather.
>
> JTT
> ----- Original Message -----
> From: "Mike Pence" <mpence <@t> grhs.net>
> To: "Annette Hall" <Annette_hall <@t> pa-ucl.com>;
> <Histonet <@t> lists.utsouthwestern.edu>
> Sent: Thursday, August 09, 2007 9:59 AM
> Subject: RE: [Histonet] Slide Staining Variability
>
>
> We have had some of these same problems and it is always been a nozzle
> or mixer problem. Do you perform regular PM's? The reaction buffer ph
> could also be off, that can cause varied staining.
>
> Mike
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Annette
> Hall
> Sent: Thursday, August 09, 2007 8:42 AM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Slide Staining Variability
>
>
> Histonetters,
>
> For those of you who use automated IHC staining systems, has anyone had
> issues with part of the slide staining and the rest of the slide not? We
> have adopted the policy of placing the control on with the patient
> tissue as a more comprehensive check of the system. However, we
> occasionally find the control is acceptable but the patient fails to
> even counterstain. This has been an intermittent problem on the Ventana
> Benchmark for some time, but the frequency has been increasing to almost
> daily.
>
> Any thoughts?
>
> Thanks,
> Annette J Hall
> Histo/Cyto/Micro Supervisor
> United Clinical Laboratories
> Dubuque, IA 52001
>
> -----Original Message-----
> From: Harrison, Sandra C. [mailto:Sandra.Harrison3 <@t> va.gov]
> Sent: Monday, August 06, 2007 3:35 PM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Leica autostainer w/integrated coverslipper
>
> Histonetters,
> Could anyone tell me if they're using the Leica AutoStainer XL (ST5010)
> with the integrated coverslipper (CV5030)? Also, can you tell me if
> you've experienced any trouble with it and how long you've been using
> it?
>
> Thanks,
> Sandy
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> End of Histonet Digest, Vol 45, Issue 13
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