Dear Emily,
I think your problems are caused by 1) excessive xylol times and
2)inadequate wax
infiltration for the thickness of the specimen.
Firstly, I would thin down your tissue to 3mm after an initial fixation of
an hour or
so. I would then shorten your processing time in xylol to 2x30 minutes.
Muscle is
particularly prone to becoming brittle and excess xylol treatment is a prime
candidate. It also comparatively dense, and thinner blocks would allow
better reagent
infiltration, particularly with wax.
Finally I would check that your wax baths were not running at too high a
temperature.

Modifications allong these lines are most likely to sort out your sectioning
problems.

Regards

Alastair McKinnon
HistoNet Server wrote:

> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 00:11:49 -0600
> From: "Donato, Elizabeth M" <edonato@fhcrc.org>
> Subject: long term paraffin section storage/tissue array
>construction/slid      e
> labeling instrumentation
>
> Hello Fellow Histonet Folks,
>
> I am trying to gather information regarding paraffin section storage
> systems, tissue array creation methods and glass slide labeling
> instrumentation.  Any knowledge that individuals would kindly be willing to
> contribute to the questions listed below would be very much appreciated.
>
> 1. I have been following the discussions of the past few weeks regarding
> long term paraffin section storage methods.  We are particularly interested
> in the method mentioned by Barry Rittman  which involved using a thin
> coating of paraffin to cover the tissue section.  Could Barry or any one
> else familiar with this method please provide me with any additional info
> regarding the technique?  How long can one store tissue sections using this
> method without compromising antigenicity?  Does this technique prevent
> oxygen from reaching the tissue?
>
> We are currently using a nitrogen storage system for storage but, for the
> large number of slides/cases we store, it is a very inefficient system
> because we are constantly going in and out of the storage boxes thereby
> intermittently exposing the sections to oxygen.  Our system consists of
> storing the paraffin sections in black slide boxes which are placed in
> double zip lock bags that are then filled with nitrogen and stored at 2-8C.
> Does anyone have a more efficient nitrogen storage system in place?
>
> 2.  We have access to a vast number of tumor paraffin blocks that are
> available to us for only a short time period.  It would be ideal to have the
> capacity to construct a tissue array or, in other words, a paraffin block
> that contains a large number (1000) of tumors.  In this way the original
> specimens would not be compromised and we would have access to a large
> source of tumors that could be characterized immunocytochemically in an
> efficient manner.  These arrays seem to be different then Battifora tissue
> rolls, which have been previously described, in that the specimens used to
> make the array are smaller in size, larger in number and arranged in a very
> organized grid like pattern.  Although, I do beleive that some of the same
> pitfalls exist in cutting and evaluating both the array and tissue rolI.  I
> have read that the National Institute of Health has a technique for
> constructing these tissue arrays using specialized instruments and have also
> heard that other groups are creating them.  Does anyone have experience with
> this process or any further information?
>
> 3.  Is anyone using an automatic slide labeler that they are happy with?  We
> would like to have the ability to, for example, input case/lab number with
> block id and maybe a date which could then be imprinted on a predetermined
> number of glass slides.  A labeler that imprints "paper" labels would also
> be considered . Vendors responses to this subject are welcome.
>
> I apologize in advance if there have already been discussions of these
> topics on the histonet.  I attempted to perform the initial research on the
> histonet archives but was unsuccessful because the archives are down and
> will not be available again until next year.  With the immense pool of
> knowledge on the histonet, I am hopeful that at least a few of my questions
> will be answered -- thanks!
>
> Liz
>
> Elizabeth Donato
> Cancer Biology
> Fred Hutchinson Cancer Research Center
> Ph:   206-667-4501
> Fax: 206-667-5815
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 01:31:09 -0600
> From: Kappeler Andreas <kappeler@patho.unibe.ch>
> Subject: Re: CD 3,4,8
>
> Hi Luis
> Among others, Pharmingen (http://www.pharmingen.com/) offers rat monoclonals
> against mouse CD3,4,8. Be aware that most of these mAbs are not suitable for
> FFPE tissues. You will have to work on fresh frozen tissues, or in some
> instances on zinc-formalin fixed material. Good luck!
> Andi Kappeler
> Institute of Pathology, University of Bern
> Switzerland
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 01:31:40 -0600
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Citrate buffer
>
> > used in antigen retrieval.  I currently use a combination of citric acid
> > and sodium citrate and recall there were some variations.  Your input
>
>   That's exactly what a citrate buffer is! Adjust the pH
>   by adding more acid to lower it, or more trisodium citrate
>   to get a higher pH. This system is effective in the range
>   3.0 to 6.2. Anything more alkaline is not a buffer, even if
>   it successfully retrieves antigens. Citrate ions may, like
>   EDTA ions, contribute to antigen retrieval by chelating calcium
>   as well as by providing a favourable pH for the reversal of some
>   of the reactions of fixation by formaldehyde. For an interesting
>   and brief exposition of this theory, read the abstract by Jasani
>   et al. in Histochemical Journal vol 29 (issue 6) p. 433.
>
>  John A. Kiernan,
>  Department of Anatomy & Cell Biology,
>  The University of Western Ontario,
>  LONDON,  Canada  N6A 5C1
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 01:32:11 -0600
> From: Kappeler Andreas <kappeler@patho.unibe.ch>
> Subject: Re: Chlamydia
>
> Hi Dana
> Two clones used to be available from the Biological Materials Distribution
> Center at the Washington Research Foundation (sorry, I have no current web
> address): clone CF-2, which is genus-specific, and clone RR402, which is
> Chlamydia pneumoniae (TWAR)-specific. We used them at dilutions of
> 1:200-1:1000 (CF-2; approx. 1-5 ug Ig/ml) and 1:50-1:250 (RR402; approx.
> 4-20 ug/ml) after antigen retrieval in 10 mM citrate buffer, pH 6.0,
> pressure cooker, StrABC-visualization. Staining is one thing, interpretation
> is the other ... Reactivity in mast cells is sometimes observed, as well as
> non specific staining of connective tissue. Good luck!
> Andi Kappeler
> Institute of Pathology, University of Bern
> Switzerland
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 03:31:11 -0600
> From: "Neuropathology" <Neuropath.Frenchay@dial.pipex.com>
> Subject: Enclosures/ Neuronal degeneration
>
> Dear All
>
> I must apologize for my error in not realising that our new software would
> send enclosures.  Linda Magraf has kindly and pleasantly explained how to
> rectify the problem.  She tells me that this is another Microsoft
> "improvement" that we could probably do without.  So I hang my head in
> shame.  My only defence is that I expect computers to do what I want with
> the minimum fuss - naive or what?
>
> The original query still applies though.  Neuronal degeneration - any help?
>
> A penitant
> Andy Shand
>
> ps Mr Bullock What do you suppose we do all day!
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 04:30:52 -0600
> From: Emma Carter <E.Carter@OxfordBioMedica.co.uk>
> Subject: Hif staining
>
> Does anyone have any protocols or suggestions for Hif staining?
>
> I want to detect hypoxia in mouse xenograft tumours, and although they
> are supposed to have had NITP in them, i cannot find any, and i _know_
> they _have_ to be hypoxic!!
>
> Hif is a long shot, but i reckon it is worth a try. Tumours are
> currently frozen and sectioned.....
>
> any help would be appreciated!
>
> emma carter
> Oxford BioMedica
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 08:02:01 -0600
> From: "McCollough, Carol" <CMCCOLLOUGH@dnr.state.md.us>
> Subject: RE: Protargol  inquiry
>
> Kathy:
>
> We purchase protargol from Cell Point Scientific, 9210 Corporate Blvd.,
> Rockville, MD 20850.  800-999-9734.  Item # 30906 (25g), CAS # 9008-42-8.
>
> Hope this helps.
> Regards -
> Carol
> *****************
> Carol B. McCollough, HT(ASCP)
> Diagnostics & Histology Laboratory Manager
> Maryland Department of Natural Resources
> Fisheries Service
> Cooperative Oxford Laboratory
> 904 S. Morris Street
> Oxford, MD 21654
>
> - -----Original Message-----
> From: kathy_hicks_at_opr-2@smtp.mcis.uchicago.edu
> [mailto:kathy_hicks_at_opr-2@smtp.mcis.uchicago.edu]
> Sent: Wednesday, December 15, 1999 6:08 PM
> To: histonet@pathology.swmed.edu
> Subject: Protargol inquiry
>
>      Can someone e-mail some information to me regarding where the best
>      place for ordering Protargol would be?  I have been told it would be
>      in Europe.
>      Thanks for any help...
>
>      Kathy Hicks
>      The University of Chicago Hospitals
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 08:46:47 -0600
> From: "MacDonald, Jennifer" <jmacdonald@sach.org>
> Subject: RE: elf bowling virus is a hoax
>
> And Elf bowling is fun!!
>
> > ----------
> > From:         ThaxtonPM@aol.com[SMTP:ThaxtonPM@aol.com]
> > Sent:         Wednesday, December 15, 1999 9:15 PM
> > To:   histonet@pathology.swmed.edu
> > Subject:      elf bowling virus is a hoax
> >
> > http://www.datafellows.com/hoaxes/elf_frog.htm
> >
> > elf bowling, frogpult, and snowball fight is safe...the above website is
> > very
> > informative about hoaxes that are circulated on the www. To be on the safe
> >
> > side, it is always best to have a virus scan program that detects trojans
> > and
> > viruses that will affect your computer. I have all three of the above
> > programs, not only do they scan as safe exe programs but the above website
> >
> > has cleared them as being "safe" as well.
> >
> >
> > okay, back in my corner now,
> >         Phyllis
> >
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 09:01:10 -0600
> From: "Margaret Gondo" <gondom@valentis.com>
> Subject: Modified Crossman's Trichrome apoptosis detection method
>
> Hi All -
>
> I got my Journal of Histotechnology the other day and it had an article in
> regarding using a modified crossman's trichrome to detect apoptosis.  Just
> wanted to know what everyone's opinion on this method was.
>
> Another treat in this month's issue is a poem by Jules Elias on pg 285.
>
> Happy Holidays everyone!
> Margaret
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 10:46:55 -0600
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Protargol  inquiry
>
> On Wed, 15 Dec 1999 kathy_hicks_at_opr-2@smtp.mcis.uchicago.edu wrote:
>
> > Can someone e-mail some information to me regarding where the best
> > place for ordering Protargol would be?  I have been told it would be
> > in Europe.
>
>   The Biological Stain Commission has been certifying silver
>   proteinates (with a preferred name of protargol-S) for about
>   20 years. Any American vendor of certified stains should have
>   an independently evaluated product in its catalog.
>
>   There are plenty of silver methods for axons that use only
>   silver nitrate and other simple, pure compounds much cheaper
>   than protargol. Some of these simpler methods are also more
>   specific for axons. (Protargol methods stain cell nuclei too.)
>
>  John A. Kiernan,
>  Department of Anatomy & Cell Biology,
>  The University of Western Ontario,
>  LONDON,  Canada  N6A 5C1
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:02:02 -0600
> From: "Keith Ryan" <KPR@wpo.nerc.ac.uk>
> Subject: RE: elf bowling virus is a hoax
>
> What I want to know is, where do you put your thumb?
>
> Keith Ryan
> Mrine Bological Asociation
> Plmouth UK
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:02:27 -0600
> From: Louise Burrell <lburrell@pathbox.wustl.edu>
> Subject: Re: Daily Digest
>
> Dear Vinnie---please contact Cathy (tech) or Dr. Roberta Sengelman who is
> a MOH'S Surgeon for info.  You can reach them through Janet (sec'y At
> (314) 747- 0239) You may use my name and good luck----also HAappiest of
> holidays to all my dear "histo-friends" till later---
>
>                                 With warmest regards,
>                                 Louise "Beezie" Burrell
>                                 Senior Technologist- Siteman Cancer Center
>                                 Tissue Procurement Core Facility Lab
>                                 Campus Box #8100; Rm # 2320; BJC North
>                                 Washington University School of Medicine
>                                 212 So Kingshighway Blvd.
>                                 St. Louis, MO.  63110
>                                 Ph:  314-454-7615
>                                 Fax: 314-454-5525
>                                 HOME # (314) 646-1188
>
> Talk to you all really soon---enjoy your families this special millenium
> holiday!!!!!!!!!!!
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:02:57 -0600
> From: Patsy.Ruegg@UCHSC.edu
> Subject: tissues and reagents needed
>
> Dear Histonetters,
>
> The NSH IHC Resource Group is in the business of helping our members find
> tissues and reagents, especially for taking the ASCP QIHC exam.
>
> Our bank is in need of some material for the next round of exam.
>
> Please help.
>
> Thank you, and Happy Holidays.
>
> Patsy Ruegg
>
> FROM THE IHC RESOURCE GROUP BANK
>
> The IHC Resource Group is once again gearing up to help support the efforts
> of applicants who have applied for the spring IHC Qualification exam.  I
> have just received a copy of the May 2000 exam, and there are a few changes
> from the last period.  The chart below describes what is being required.
>
> BOARD OF REGISTRY EXAN FOR IMMUNOHISTOCHEMISTRY QUALIFICATION
>
> MAY 2000
>
> Project #       Antibody(ies)   Tissue
> 1       HMB 45  Melanoma
> 2       LCA     Normal tonsil or lymph node
> 3       LMW & HMW keratin       Prostate
> 4       Chromogranin    Stomach
> 5       Estrogen receptor       Breast CA
> 6       HPV     Cervix
> 7       T-cells & B-cells       Frozen lymph node or tonsil
> 8       Factor VIII related antigen     Kidney w cortex & medulla
> 9       Muscle specific antigen Tx both pos and neg for the antibody
>
> The bank is very low on some tissues, on probe and probe detection systems.
> I am earnestly asking for your help.  We are out of probe and probe kits,
> and as you can see, the applicants will now have to perform HPV on infected
> cervix.  We have no tissue or reagents presently to support this part of the
> exam.  If anyone can donate anything, tissue being in the greatest demand,
> please let me know.  Ask your vendors for samples for donation to this
> effort.  This might help us further.
>
> Additionally we are low on prostate, ER pos breast CA, melanoma, and kidney
> tissue that has both medulla and cortex, as well as frozen lymph node or
> frozen tonsil.
>
> The antibody stores are pretty good.  If anyone can contribute a paraffin
> block for any of the above tissue, please email me at
> emacrea@ventanamed.com, call me at 800-227-2155 ext 2739, or simply mail
> blocks to Ethel R Macrea
>
> 4740 N Brookeview Dr
> Tucson, AZ 85704
> Or
> C/o Ventana Medical Systems Inc.
> 3865 N Business Center Dr
> Tucson, AZ 85705
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:42:30 -0600
> From: Gayle Callis <uvsgc@msu.oscs.montana.edu>
> Subject: zinc formalin fixation or zinc fixative?
>
> A comment about zinc formalin fixation for these murine cell surface
> markers.  Andi is correct about the fact the these, particularly the CD4
> and CD8 markers may  not stain after FFPE prepared tissues.
>
> Caveat:
>
> Pharmingen does not tout zinc formalin fixed tissue, they recommend
> (in their catalog with recipe and how to fix) a zinc fixative which does
> NOT contain formalin. It is TRIS buffered Zinc fixative, containing zinc
> chloride, zinc acetate and calcium acetate.  I think their use of the
> terminology Zinc Fixative is easily confused with zinc formalin which we
> are so familiar with.  Kind of wish they would call it ZincTRIS or
> at least nonformalin zinc fixative, sorry just my hangup, but far less
> confusing.  This fixative will work with these cell marker antibodies,
> per publication by Nitta et al, in Cell Vision 1:73, 1997. Alk phos substrate
> method must be used, as you cannot rid tissue of endogenous peroxidase after
> the ZincTris fixation.  This fixative also eliminates the need for
> antigen retrieval IF you make sure the tissue is 100% ZnTris fixed,
> the alcohols in processing continue the fixation, and
> I am aware of one person who had to retrieve because of this reason.
> Somewhat defeated one  purpose of this nice fixative!
>
> PharMingen does give a NBF fixative, and maybe zinc formalin would work,
> with extensive antigen retrieval, but it has not worked for many others with
> these CD markers. I was hoping to point out their recommendation of this
> formalin free zinc fixative and alleviate confusion before one launches
> into a huge mouse tissue  project with the wrong fixative.
>
> Some other things learned about ZnTris fixative, is some tissues make take
> less time to fix, others longer than publication recommendation, so a
> fixation time study might be in order to optimize fixation first.
>
> Sorry to be a Bah, Humbug, but hope this helps avoid confusion.
> We prefer to do frozens, acetone fixed but have had success with ZnTris,
> formalin of any kind is avoided for murine CD markers
>
> I would like to know if anyone has ever had good success with formalin based
> fixatives with murine CD3, 4, and 8.
>
> What a long lecture!
> Gayle Callis
>
>         ********************************************************
>
> Hi Luis
> Among others, Pharmingen (http://www.pharmingen.com/) offers rat monoclonals
> against mouse CD3,4,8. Be aware that most of these mAbs are not suitable for
> FFPE tissues. You will have to work on fresh frozen tissues, or in some
> instances on zinc-formalin fixed material. Good luck!
> Andi Kappeler
> Institute of Pathology, University of Bern
> Switzerland
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:43:40 -0600
> From: Shelley Sheridan <sksherid@yahoo.com>
> Subject: gallyas stain
>
> Hello all.
> I am working to develop the Gallyas stain for
> neuropathology.  We normally use immunohistochemistry
> for our diagnostics, so we have very little experience
> with special stains.  We are having problems with the
> developer reaction.  We know that when you add
> together the two developer solutions the mixture will
> turn cloudy and then go clear.  Our solution is
> staying cloudy, even though we use acid washed
> glassware and very clean water.  The cloudy mixture is
> developing the sections, but the staining is not
> ideal.  We would like to get the developer reacting
> correctly before we start using the stain on more
> important tissue.  Does anyone have any ideas?  How
> slowly do you add the solutions together?  Do you use
> a separation funnel?  Is there some special technique
> to making the developer that we don't know about?
> Thank you ahead of time for your help.
>
>
>
> =====
> Shelley K. Sheridan
> Research Specialist
> Center for Neurodegenerative Disease Research
> University of Pennsylvania Medical School
> Philadelphia, PA 19104
> Phone:(215)614-0051
> __________________________________________________
> Do You Yahoo!?
> Thousands of Stores.  Millions of Products.  All in one place.
> Yahoo! Shopping: http://shopping.yahoo.com
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:44:09 -0600
> From: Cynthia Favara <cfavara@niaid.nih.gov>
> Subject: RE: CD 3,4,8
>
> Luis,
>         Try R&D Research and PharMingen they both ahve web sites I think.
> Cynthia Favara
> NIAID/RML/LPVD
> 903 South 4th Street
> Hamilton, MT 59840
> PH: 406-363-9317
> FAX: 406-363-9286
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:44:56 -0600
> From: Cynthia Favara <cfavara@niaid.nih.gov>
> Subject: RE: Removal of DAB Chromogen
>
> I am not familiar with these antibodies so have a few questions. Is the
> staining seen in the same cells? If not I would just do a double stain with
> a different substrate say VIP or AEC. I have done double staining using 2
> HRP reactions and any residual HRP is removed by a second peroxidase block
> and or a second HIER. I suggest this as I know it works in my lab. Other
> suggestion would be to use a mild bleach solution which should also remove
> the DAB, but I don't know what other efects it may have. Good luck!! and
> Happy Holidays!!!
> Cynthia Favara
> NIAID/RML/LPVD
> 903 South 4th Street
> Hamilton, MT 59840
> PH: 406-363-9317
> FAX: 406-363-9286
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 11:45:39 -0600
> From: "Terrett, Barb" <Barb.Terrett@uhn.on.ca>
> Subject: microwaving
>
> Hi there,
> Would anyone be able to give me a reference or a website where I can learn
> exactly what microwaving is doing to the tissue and how microwaving works?
> The question was raised here, and I only give a very general answer to it,
> so now my curiosity is peaked.What is really happening?
> Thanks very much
> b
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 12:54:03 -0600
> From: "Peter A. Takes" <ptakes@stereotaxis.com>
> Subject: Re: microwaving
>
> Kok & Boon. Microwave Cookbook for Microscopists. Art and Science of
> Visualization. Third revised edition. Coulomb Press Leyden, Leiden,
> Netherlands. 1992.
>
> - --
> Peter A. Takes, Ph.D., RAC
> Director, Clinical & Regulatory Affairs
> STEREOTAXIS, Inc.
> Ph. 1-314-615-6964; Pager 1-314-841-9351
> ptakes@stereotaxis.com
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 12:54:46 -0600
> From: Katri Tuomala <katri@istar.ca>
> Subject: Re: amplification kit
>
> Hi Klazien,
> You may have received the answer to your inquiry already, but I have not
> seen it. I have used this kit on paraffin sections with antibodies that
> are normally not demonstrable in paraffins. I had to do a lot of
> tweaking with the antigen retrieval and antibody dilutions, because of
> the background staining. I have a feeling you may be successful with a
> much higher primary antibody dilution. Do a wide titration first to get
> closer to real titre and then narrow it down. Good luck!
>
> Katri
>
> Katri Tuomala
> Anatomic Pathology
> St.Joseph's Hospital
> Hamilton, Ontario, Canada
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 12:55:12 -0600
> From: Mary Reischl <Mary.Reischl@dakousa.com>
> Subject: Job opportunity: Technical Service Rep for DAKO Corporation
>
> DAKO is an international biomedical company that develops and markets
> immunological reagents and diagnostic kits for use in clinical and research
> laboratories.
>
> We are looking for a full time, dependable individual (actually two) to
> perform installations and training for the DAKO Autostainer and reagents,
> working on-site at DAKO's customers' locations.  To assure customer
> satisfaction, the candidate will assist the laboratories in the development
> of  applications of chemistries on the Autostainer and be available for
> frontline troubleshooting and problem solving, including light repairs on
> the Autostainers, in labs using DAKO products.
>
> The candidate will also assist the sales organizations in technical
> presentations and demonstrations for DAKO customers.  There will be
> extensive travel in the territory.  Two new territories are available.  The
> Northwestern/Upper Mid Western territory can be based in the San Francisco
> Bay Area, In Stockton, CA or Sacramento, CA.  Seattle is also possible.  The
> Texas territory, including Oklahoma, Arkansas, Louisiana and Mississippi,
> can be based in the Dallas/Ft. Worth, Houston or San Antonio areas.  Chicago
> as a base is also a possibility.
>
> Previous experience in histology and automated immunohistochemistry is
> required.
>
> Please respond to:
>         DAKO Corporation
>         6392 Via Real
>         Carpinteria, CA 93013
>         employment@dakousa.com
>         Fax (805) 566-5419
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 13:34:55 -0600
> From: Patsy.Ruegg@UCHSC.edu
> Subject: web site access
>
> we have been having trouble with the domain for our State, Region VII, and
> IHC web site, we hope to have the problem fixed in a few days but for now
> the http://coloradohisto.org site can be accessed by going to:
> <http://histo.nocontrol.net>
> Patsy Ruegg
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 13:36:42 -0600
> From: "Terrett, Barb" <Barb.Terrett@uhn.on.ca>
> Subject: microwaving
>
> Thanks to everyone for suggesting the Microwave Cookbook. I was able to find
> an old copy of it in our histo. lab.
> b
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 13:37:07 -0600
> From: "Baileyhealy, Irene {Immu~Palo Alto}" <IRENE.BAILEYHEALY@ROCHE.COM>
> Subject: Un-subscribe
>
> Thank you
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 13:37:40 -0600
> From: "Slap, Steven" <SSlap@ebsciences.com>
> Subject: RE: microwaving
>
> Hi HistoNetters!
>
> I would still recommend Kok and Boon's Microwave Cookbook for Microscopists
> as the most thorough text describing the effects of microwaving on
> histological procedures.  A second good reference would be Gary Login's
> Microwave Tool Book.
>
> I would be happy to try to address anyone's questions either on the list or
> off, and there are both commercial and general reference materials on
> microwaving available at our web site (http://www.ebsciences.com)
> <http://www.ebsciences.com)> .
>
> Best regards,
> Steven E. Slap, Vice-President
> Energy Beam Sciences, Inc.
> "The Laboratory Microwave Company"*
>
>         -----Original Message-----
>         From:   Terrett, Barb [SMTP:Barb.Terrett@uhn.on.ca]
>         Sent:   Thursday, December 16, 1999 12:30 PM
>         To:     'histonet@pathology.swmed.edu'
>         Subject:        microwaving
>
>         Hi there,
>         Would anyone be able to give me a reference or a website where I can
> learn
>         exactly what microwaving is doing to the tissue and how microwaving
> works?
>         The question was raised here, and I only give a very general answer
> to it,
>         so now my curiosity is peaked.What is really happening?
>         Thanks very much
>         b
>
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 16:06:04 -0600
> From: Mary Ann Miller <millerma@email.uc.edu>
> Subject: Re: long term paraffin section storage/tissue array
> construction/slide labeling instrumentation
>
> Dear Liz, and others
>
> Our lab also works with hundreds and hundreds of different tumors since we
> are the repository for several SWOG GI protocols. We have attempted to deal
> with these issues for many years. We set up an experiment in 1996 whereby
> we studied several different tumors known to have differing intensities of
> staining on freshly cut slides. Tissues were cut on Evergreen ++ slides and
> then studied at monthly intervals for 2 years. We routinely cut our tissues
> using a 42C waterbath containing no additives. We then allow the slides to
> dry at RT for at least 24 hours before stacking and filing for later use.
> For this study we coated half the slides with paraffin and left the other
> half plain.The results were variable but mostly block dependent.(We had no
> knowledge of fixation techniques since the blocks came from 50 different
> places.)  The slides coated with paraffin actually lost far more activity
> over time than those simply stored at RT in old Shandon filter card boxes.
> They also took up much more storage space. We abandoned this method. The
> antibodies we specifically studied for this experiment were P-53, WAF-1 and
> Ki67. In addition we have checked many other antibodies over time. We have
> found our results to be dependent on how carefully the slides were
> originally cut and on the individual antibody.(could also be the antigen of
> course). A good number of the antibodies we use seem to remain stable over
> at least several years. For the most part staining intensity may go from 4+
> to 3+ but is still quite acceptable. The problem is is of course worse if
> the initial stain is only 2+. One other thing I should also note,  we use
> the Ventana ES Immunostainer.
>
> I'm not quite sure what you mean by tumor arrays but we routinely make up
> what we call our tumor sausage blocks. For these we take small (2-3mm)
> pieces of "old" tumors from our surgical files, melt them down and reembed
> 8-10 of these pieces in one larger block. We have colon, breast, lung
> sausages etc. This number is plenty to show variability across tissues. We
> then cut 10-20 slides at a time and store them as above for use as positive
> controls.
>
> One more thing --if you receive replies directed only to you could you
> please share them with me? I hate to miss important information.
>
> Sincerely,    Mary Ann
>
>
>
> At 09:08 PM 12/15/99 -0800, you wrote:
> >
> >Hello Fellow Histonet Folks,
> >
> >I am trying to gather information regarding paraffin section storage
> >systems, tissue array creation methods and glass slide labeling
> >instrumentation.  Any knowledge that individuals would kindly be willing to
> >contribute to the questions listed below would be very much appreciated.
> >
> >1. I have been following the discussions of the past few weeks regarding
> >long term paraffin section storage methods.  We are particularly interested
> >in the method mentioned by Barry Rittman  which involved using a thin
> >coating of paraffin to cover the tissue section.  Could Barry or any one
> >else familiar with this method please provide me with any additional info
> >regarding the technique?  How long can one store tissue sections using this
> >method without compromising antigenicity?  Does this technique prevent
> >oxygen from reaching the tissue?
> >
> >We are currently using a nitrogen storage system for storage but, for the
> >large number of slides/cases we store, it is a very inefficient system
> >because we are constantly going in and out of the storage boxes thereby
> >intermittently exposing the sections to oxygen.  Our system consists of
> >storing the paraffin sections in black slide boxes which are placed in
> >double zip lock bags that are then filled with nitrogen and stored at 2-8C.
> >Does anyone have a more efficient nitrogen storage system in place?
> >
> >2.  We have access to a vast number of tumor paraffin blocks that are
> >available to us for only a short time period.  It would be ideal to have the
> >capacity to construct a tissue array or, in other words, a paraffin block
> >that contains a large number (1000) of tumors.  In this way the original
> >specimens would not be compromised and we would have access to a large
> >source of tumors that could be characterized immunocytochemically in an
> >efficient manner.  These arrays seem to be different then Battifora tissue
> >rolls, which have been previously described, in that the specimens used to
> >make the array are smaller in size, larger in number and arranged in a very
> >organized grid like pattern.  Although, I do beleive that some of the same
> >pitfalls exist in cutting and evaluating both the array and tissue rolI.  I
> >have read that the National Institute of Health has a technique for
> >constructing these tissue arrays using specialized instruments and have also
> >heard that other groups are creating them.  Does anyone have experience with
> >this process or any further information?
> >
> >3.  Is anyone using an automatic slide labeler that they are happy with?  We
> >would like to have the ability to, for example, input case/lab number with
> >block id and maybe a date which could then be imprinted on a predetermined
> >number of glass slides.  A labeler that imprints "paper" labels would also
> >be considered . Vendors responses to this subject are welcome.
> >
> >I apologize in advance if there have already been discussions of these
> >topics on the histonet.  I attempted to perform the initial research on the
> >histonet archives but was unsuccessful because the archives are down and
> >will not be available again until next year.  With the immense pool of
> >knowledge on the histonet, I am hopeful that at least a few of my questions
> >will be answered -- thanks!
> >
> >Liz
> >
> >Elizabeth Donato
> >Cancer Biology
> >Fred Hutchinson Cancer Research Center
> >Ph:   206-667-4501
> >Fax: 206-667-5815
> >
> Mary Ann Miller
> University of Cincinnati
> College of Medicine  ML 529
> 231 Bethesda Avenue
> Cincinnati, Ohio  45267
> FAX: (513) 558-2289
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 16:20:31 -0600
> From: "Yandl, Emily" <Emily.Yandl@genzyme.com>
> Subject: processing rat muscle
>
> Hi all!
>
> I'm hoping someone can help me with this problem.  Our lab is working on a
> project for a researcher who is looking at rat sciatic nerve along with the
> associated muscle.  So far we have tried two different processing procedures
> with less than satisfactory results.  Following both processing procedures,
> upon cutting, the muscle appears very glassy and yellowish, like amber.
> After the longer cycle, full sections were able to be cut after soaking, but
> the sections exploded after being placed on the water bath.  After the
> shorter cycle, the muscles held together better on the waterbath, however
> developed ragged holes in the center of the tissue.  The pieces of muscle
> are
> approx. 2cm x 1.5 cm x 0.6 cm in dimension, and were processed on a Shandon
> Citadel 2000 processor.
>
> If anyone has any processing schedules that work for them or ideas of why
> this isn't working, I would love to hear your comments.  The processing
> schedules used are listed below.
>
> The longer schedule is:
> at least 48 hrs. 10% NBF fixation
> 2--30 min changes PBS
> 1--1hr 70% Alc
> 2--1hr changes 90% Alc
> 2--1hr changes 100% Alc
> 3--1.5 hrs changes in Xylene
> 2--2 hr changes in paraffin
>
> The shorter schedule is:
> at least 48 hrs in 10% NBF fixation
> 2--30 min changes in PBS
> 1--45 min change in 70% Alc
> 2--45 min changes in 90% Alc
> 2--45 min changes in 100% Alc
> 3--45 min changes in Xylene
> 2--2 hr changes in paraffin.
>
> Thanks in advance for any help,
>
> Emily Yandl
> Pre-Clinical Biology
> Genzyme Corp.
> 1 Mountain Rd.
> Framingham, MA  01701
> (508) 872-8400 x23623
> emily.yandl@genzyme.com
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 17:31:24 -0600
> From: happydays <happydays@mciworld.com>
> Subject: IHC Exam
>
> Hello;
> I am interested in taking the NSH IHC Exam and would like to know where
> I can get the information on the exam?
>
> Thanks
> Penny Fritz
> York Hospital
> happydays@mciworld.com
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 17:32:33 -0600
> From: "Hagerty, Marjorie A." <mhagerty@emc.org>
> Subject: BILLING INQUIRY
>
> Can anyone point me in the right direction? I need some documentation, if in
> fact there is any. It is my understanding that if you bill for a special
> stain it must be mentioned in the report. Some here feel it is OK to just
> state generically that specials were done. Need to find out what is the
> right way to do it.
>
> Anyone know of any specific regulations and where I can get the
> documentation? Thanks in advance for any help.
>
> Marg
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 17:59:10 -0600
> From: "Baileyhealy, Irene {Immu~Palo Alto}" <IRENE.BAILEYHEALY@ROCHE.COM>
> Subject: FW: Unsubscribe
>
> > Thank you
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 18:20:45 -0600
> From: larisonk@uoneuro.uoregon.edu
> Subject: Re: amplification kit
>
> If I remember correctly, Mr Bosch was trying to use biotinylated tyramides to
> amplify
> signals in frozen sections.  I haven't had any experience with the
> biotinylated
> tyramides, but background is definitely a problem in frozen sections when I
> use the
> fluorescein tyramides.  Titrating the primary antibody doesn't help.  Oddly
> enough,
> if I use the fluorescein tyramide on whole embryos, I get gorgeous staining
> with
> virtually no background.  Is the substrate sticking to the cut membranes?
> It's a
> mystery to me!
>
> Karen in Oregon
>
> Date:          Thu, 16 Dec 1999 12:43:58 -0500
> From:          Katri Tuomala <katri@istar.ca>
> Subject:       Re: amplification kit
> To:            Klazien Bosch <k.s.bosch@amc.uva.nl>
> Cc:            HistoNet@pathology.swmed.edu
>
> Hi Klazien,
> You may have received the answer to your inquiry already, but I have not
> seen it. I have used this kit on paraffin sections with antibodies that
> are normally not demonstrable in paraffins. I had to do a lot of
> tweaking with the antigen retrieval and antibody dilutions, because of
> the background staining. I have a feeling you may be successful with a
> much higher primary antibody dilution. Do a wide titration first to get
> closer to real titre and then narrow it down. Good luck!
>
> Katri
>
> Katri Tuomala
> Anatomic Pathology
> St.Joseph's Hospital
> Hamilton, Ontario, Canada
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 21:31:52 -0600
> From: RHD101@aol.com
> Subject: Re: BILLING INQUIRY
>
> By special stain I take it you mean immunohistochemistry. It should be
> documented in the surgical pathology report for several reasons, including:
>
> 1. The Association of Directors of Anatomic and Surgical Pathology recommend
> it. (http://www.panix.com/~adasp/imstain.htm)
>
> 2. Medicare may require it for reimbursement, but I am not sure about that
> one.
>
> 3. If you are inspected by CAP, they might get ya, depending on the
> inspector. See below.
>
> QUESTION: 08:1240 (ANP.09039) PHASE: II
> #160#
> Is there a mechanism to correlate the results of specialized studies (e.g.,
> electron microscopy, immunohistochemistry, nucleic acid probes, cytogenetics)
> with the morphologic diagnosis?
> #160#
> COMMENTARY: 08:1240 (ANP.09039) PHASE: II
> #160#
> THE SURGICAL PATHOLOGY SECTION LACKS A MECHANISM TO CORRELATE THE RESULTS OF
> SPECIALIZED STUDIES (e.g., ELECTRON MICROSCOPY, IMMUNOHISTOCHEMISTRY, NUCLEIC
> ACID PROBES, CYTOGENETICS) WITH THE MORPHOLOGIC DIAGNOSIS. IT IS NOT IN THE
> BEST INTERESTS OF THE PATIENT TO HAVE POTENTIALLY CONFLICTING DIAGNOSES OR
> INTERPRETATIONS RENDERED BY DIFFERENT SECTIONS OF THE LABORATORY. THE
> PATHOLOGIST SHOULD CORRELATE ALL OF THE SPECIAL STUDIES, RECONCILE
> CONFLICTING DATA, AND RENDER A FINAL INTERPRETATION OF ALL CORRELATED
>STUDIES.
> #160#
> REFERENCE: EDITORIAL. INCORPORATION OF IMMUNOSTAINING DATA IN ANATOMIC
> PATHOLOGY REPORTS. AM J CLIN PATHOL. 1993;99:1.
>
> Hope this helps.
>
> John Shannon, MD
>
> ----------------------------------------------------------------------
>
> Date: 16 Dec 1999 23:00:57 -0600
> From: Immunohisto@aol.com
> Subject: PAS Alcian Blue 2.5
>
> I am looking for a good procedure for the PAS Alcian Blue 2.5 and what kindof
> control to use to get the best results. Any help would be appreciated.
>
> Thank you,
> Susan Blanche
>
> Here are the messages received yesterday!