----- Original Message -----
From: "HistoNet Server" <histonet@pathology.swmed.edu>
To: "HistoNet Server" <histonet@pathology.swmed.edu>
Sent: Friday, December 24, 1999 12:11 AM
Subject: Daily Digest


>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 04:45:23 -0600
> From: "Sabrina Dize" <Dize_Sabrina@piedmont.promina.org>
> Subject: Merry Christmas and Thank you
>
> Hi all!
>
> I just wanted to let you all know how much it means to me to know that
there
> are people out there who care about me and my family. This past summer
when
> everyone sent Morgan cards and gifts it did indeed lift my heart.
>
> Thank you
>  for riding through the rough waters
>  of changing with me.
> Thank you for holding my hand.
> And thank you for waiting.
> Thank you
> for taking the time to help me.
> Thank you for standing beside me.
> Thank you for each day
> you were there for me...
>
> thank you for being there for me
> when I needed you most.
>
> I also want to wish everyone a most wonderful Christmas ( or I hope your
> Hanukkah was a good one).
>
> Lynn, Morgan's Mom
>
> Please feel free to contact us!
> ldize@mindspring.com
> Dize_Sabrina@piedmont.promina.org
> (there is an underscore between my first and last name)
> Dize
> 2666 Bluebird Circle
> Duluth, GA., 30096
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 06:46:02 -0600
> From: "Kellar, Eric" <kellarec@MSX.UPMC.EDU>
> Subject: Season's Greetings
>
> To all HistoNet friends and colleagues around the world, regardless of
> whether you are celebrating the Winter Solstice, Chanukah, Christmas,
> Kwanzaa, the beginning of Ramadan, or some other special day, I wish you
and
> your family the best of holidays as we celebrate this time of year. There
is
> no better gift to share than that of knowledge and experience. Thanks to
> all!
>
>
> Eric C. Kellar
> Pittsburgh, PA
>
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 07:15:35 -0600
> From: "Mike Kirby" <mikek@mail.saimr.wits.ac.za>
> Subject: Tissue samples "Ethics"
>
>
>
> Thanks Russ, now that you put some "meat" on the "bones", the Ethics part
of
> Histopathology tissue samples becomes a little more evident to my
generally
> befuddled, Haematology orientated brain.
> I remember the "Bristol" case very well, and I suppose that it was a
rather
> heart wrenching time for the parents involved, especially when you
discover
> that bits and pieces of your child are floating around in a bottle.
> It has always been an "unwritten contract" between the surgeon/clinician
> and the patient that any tissue sample/organ was removed for diagnostic
> purposes, or because it was diseased , and what became of it after that,
was
> best left to the Histopathology Lab.
> Now that the general public has become much more knowledgeable of the
> medical world and their rights, I can foresee, as you say, more and more
> "problems" associated with tissue samples.
> It all boils down to the question of "who's tissue/organ is it anyway"
once
> it has been removed from the body. Does it in theory, still belongs to the
> patient (can a blood donor ask for his "pint" back?) or is it the property
> of the Histopathology Lab to do with as they see fit?
> This is a whole 44 gallon drum of worms just waiting to be opened.
> Lets have some input from the rest of you salami slicers.
>
> Mr.M.Kirby
> Johannesburg
> South Africa.
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 08:46:07 -0600
> From: "Instrumedics, Inc." <cfss@idt.net>
> Subject: Greetings
>
> To all Histonetters,
> Instrumedics wishes you all very Happy Holidays and a New Millennium that
is
> kinder and gentler!
>
> We, as a vendor, also want to express our deep appreciation for your
> helpfulness and interest in learning about what we offer for the
advancement
> of the practice of histology.
>
> Thanks!
>
> Bernice
> schiller@instrumedics.com
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 10:45:12 -0600
> From: thaxtonpm@aol.com
> Subject: You have a virtual greeting card number 2308314150176 waiting for
> you!
>
> Hi Histonet,
>
> Phyllis Thaxton stopped by our site, Free Web Cards by Webmania Designs,
and
> created a Virtual Card just for you!
>
> You may pick it up from our card pick-up window located at:
>
>
>  http://freewebcards.com
>
>  Your card number is: 2308314150176
>  (enter this number in the space provided under "Pickup Card" in the menu
on
> left and press the "View" button)
>
> Alternatively you can pick it up by clicking on the link below (For AOL
mail,
> use directions below):
>
>
> http://www.webmaniadesigns.com/cgi-bin/magiccard.cgi?2308314150176
>
> ****AOL OR HTML enriched e-mail program Users****
>
> If you are using AOL mail, <a
> href="http://www.webmaniadesigns.com/cgi-bin/magiccard.cgi?2308314150176
> ">just click here</a> to pick-up your card.
>
> **********IMPORTANT**********
>
> The card will remain on our server for 21 days after you've picked the
card up
> and 30 days for cards that have not been retrieved. ***DIRECTIONS FOR
SAVING
> THE CARD TO YOUR HARD DRIVE FOR PERMANENT VIEWING CAN BE FOUND AT:
> http://freewebcards.com/save.shtml. We recommend that you save your card
the
> first time you view it.
>
> If you are having problems retrieving your card, please go to
> http://webmaniadesigns.com/help.html and read the help section. If you are
> still experiencing difficulties, please fill out the form provided on that
> page. Thank you
>
> If you are having problems going to my help page, please send an e-mail to
> cardmaster@webmaniadesigns.com be sure to include your card number and any
> error message you are receiving as well as the type of Web browser (and
the
> version if possible) you are using, i.e. Netscape, Internet Explorer, AOL,
> etc. Thank you.
>
>
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 11:01:17 -0600
> From: "Bennett, Catherine (Katie)" <cbennett@lrri.org>
> Subject: Antigen Retrieval
>
> I realize not many histonetters may be online right now, but to those out
> there, I present my current dilemma.
>
> I am trying to do double immuno staining for BrdU and Vimentin in 10%
> neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> labeling with almost all of the microwave antigen retrieval procedures
I've
> been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> EDTA).  But the Vimentin stain after microwave AR is really weak.  Without
> antigen retrieval, I get no BrdU staining but awesome Vimentin stains.  My
> protocol always includes a 5 minute 0.05% Protease K enzyme digestion
step,
> and a 1h HCL denaturation step.  Without the microwave AR, these alone
won't
> do the trick for the BrdU.
>
> I'm guessing my next step is to ditch the microwave AR since it kills the
> vimentin and play around with various enzyme digestion times to boost the
> BrdU.
>
> Anyone have any insight or suggestions?
>
>
>
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 11:31:15 -0600
> From: Amos Brooks <atbrooks@snet.net>
> Subject: Re: ER/PR, Her2 Regulatory Questions
>
> Hi Louri,
>     We do ALOT of Herceptest at our lab (between 20 and 40 each day). One
of
> the
> marvels of this test and its stiff regulations about target retrieval is
that
> it
> leaves very little room for variation. You don't (or can't) change the
titer,
> incubation times or change the retrieval technique. This in it's own
> complexity
> makes valiation and optimization simple for the techs.
>     Know in your heart there will always be left over reagent. It is not
like
> assembling a bookshelf and finding extra screws (been there done that).
Your
> leftover reagent is still usable next time as long as it is the same lot
> number.
>
>     The only people who cant run the test are those who cannot follow the
time
> constraints to a "T". Reproducibility is the key.
>     Another comment worthy of note is that you may not need to run a
control
> with each case. A control for each run (48 slides) should be sufficient. A
> control for each case would burn through all our controls too quick. We
run
> two
> slides per test, one with the primary and one with the enclosed negative
> control
> reagent. Then for each staining run a positive and negative control and
the
> enclosed control slide from DAKO.
>     Our reps have agreed this is sufficient for the regulations, but extra
> work
> is never frowned upon by inspectors.
> Happy Holidays
> Amos Brooks
>
> Louri Caldwell wrote:
>
> > Hello everyone,
> >
> > I have been asked to initiate immunoperoxidase procedures for ER/PR and
Her2
> > on breast cancer cases and I want to be sure we follow all applicable
> > regulations.  I have read the CAP, FDA, and CLIA guidelines, but I want
to
> > be sure I've done everything correctly.
> >
> > Our current procedure is as follows:  We use Dako's ER & PR primary
> > antibodies with their LSAB2 kit for ER & PR, and the Herceptest kit for
> > Her2.  The lot of each antibody and kit is tracked as to date
> > ordered/received/opened/discarded, along with the initial reactivity of
each
> > antibody/kit, and the cases tested using each lot.  Along with each case
> > tested, a control block is being used that has a range of known
reactivity
> > to each antibody (from 0 to 3+) and a negative control section of the
case
> > being tested.  In addition to these 2 controls, the Herceptest kit
control
> > slide is run with the Her2.  The results of each stain as dictated by
the
> > pathologist is also tracked.
> >
> > Here are the questions I have:
> >
> >   Are there any additional controls that need to be run or validation
> > procedures I need to go through in order to be compliant?
> >
> >   Do I need to go through separate validation procedures if I use less
> > reagent than the Herceptest instructions dictate?  If so, to what
extent?
> >
> >   Are there any restrictions as to who can perform these procedures?
> >
> > Any assistance would be greatly appreciated.  Wishing all a very safe
and
> > happy holiday season.
> >
> > Louri Caldwell
> > ______________________________________________________
> > Get Your Private, Free Email at http://www.hotmail.com
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 11:45:53 -0600
> From: Lynn Gardner <lynn-gardner@uiowa.edu>
> Subject: Cells
>
> Dear Histonetters,
>
> First of all Merry Christmas and Happy New Year to all!! I have a question
> that hopefully someone will have an answer.
>
> We have grown up some cells in a petri dish. We are now wanting to try and
> process these cells, however, we need to keep them in their shape and not
> disrupt them. Does anyone have an idea of how to do this or reference to
> journal articles that would contain this information?
>
> I thought we could place the cells in agar and then process them but don't
> know what type of agar would process and cut well. Would like any imput
> possible.
>
> Also, is there anyone out there that knows of a cytology network like our
> histonet?
>
> Thanks for your help everyone! Happy Holidays!
>
> Sincerely,
> Lynn Gardner
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:02:15 -0600
> From: Amos Brooks <atbrooks@snet.net>
> Subject: Re: Antigen Retrieval
>
> Hi,
>     Proteinase K (PK) is an enzyme digestion meant to take the place of
heat
> induced epitope retrieval (HIER). For many antibodies this works great.
But
> you
> shouldn't do both. Try the Vimentin using HIER and no PK or vice versa.
> Remember
> it may be that you may need to use HIER on one and PK on the other both do
not
> need to be run the same since they are different antibodies.
> Amos Brooks
>
> "Bennett, Catherine (Katie)" wrote:
>
> > I realize not many histonetters may be online right now, but to those
out
> > there, I present my current dilemma.
> >
> > I am trying to do double immuno staining for BrdU and Vimentin in 10%
> > neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> > labeling with almost all of the microwave antigen retrieval procedures
I've
> > been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> > EDTA).  But the Vimentin stain after microwave AR is really weak.
Without
> > antigen retrieval, I get no BrdU staining but awesome Vimentin stains.
My
> > protocol always includes a 5 minute 0.05% Protease K enzyme digestion
step,
> > and a 1h HCL denaturation step.  Without the microwave AR, these alone
won't
> > do the trick for the BrdU.
> >
> > I'm guessing my next step is to ditch the microwave AR since it kills
the
> > vimentin and play around with various enzyme digestion times to boost
the
> > BrdU.
> >
> > Anyone have any insight or suggestions?
> >
> > ****************************
> > Catherine "Katie" Bresee Bennett
> > Sr. Technical Associate
> > Lovelace Respiratory Research Institute
> > Albuquerque, New Mexico
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:02:55 -0600
> From: "Genty Family" <lcjx@pdq.net>
> Subject: Re: Antigen Retrieval
>
> Hi Catherine:
>
> It has been my experience that Vimentin is actually enhanced by Heat
induced
> AR with 0.01M citrate buffer pH 6.0.  However I do not know the effects of
> the protease and the HCl on Vimentin antigenicity.  Perharps some of our
> histonet gurus can provide some insight.
>
> When performing Brdu stains, I used to use a similar procedure, AR with
> 0.01M citrate buffer followed by an 60 min  RTemp incubation in 2N HCl
then
> by a standard HRP-labeled streptavidin technique.  2N HCL times may be
lower
> these days.
>
> You may want to try this, in a worst case scenario the antigenicity may be
> affected by the HCl.  But even if that were the case you could reverse the
> order and adjust your titers and detection systems accordingly.
>
> I have not kept up with the literature, but there may be even simpler
> approaches to Brdu IHC that may better preserve your Vimentin
antigenicity.
>
> I hope this is somewhat helpful.
>
> Carlos Genty
> Breast Cancer Center
> Baylor College of Medicine
> Houston, Texas
> - ----- Original Message -----
> From: Bennett, Catherine (Katie) <cbennett@lrri.org>
> To: 'Histonet Server' <histonet@pathology.swmed.edu>
> Sent: Thursday, December 23, 1999 10:51 AM
> Subject: Antigen Retrieval
>
>
> > I realize not many histonetters may be online right now, but to those
out
> > there, I present my current dilemma.
> >
> > I am trying to do double immuno staining for BrdU and Vimentin in 10%
> > neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> > labeling with almost all of the microwave antigen retrieval procedures
> I've
> > been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> > EDTA).  But the Vimentin stain after microwave AR is really weak.
Without
> > antigen retrieval, I get no BrdU staining but awesome Vimentin stains.
My
> > protocol always includes a 5 minute 0.05% Protease K enzyme digestion
> step,
> > and a 1h HCL denaturation step.  Without the microwave AR, these alone
> won't
> > do the trick for the BrdU.
> >
> > I'm guessing my next step is to ditch the microwave AR since it kills
the
> > vimentin and play around with various enzyme digestion times to boost
the
> > BrdU.
> >
> > Anyone have any insight or suggestions?
> >
> >
> >
> > ****************************
> > Catherine "Katie" Bresee Bennett
> > Sr. Technical Associate
> > Lovelace Respiratory Research Institute
> > Albuquerque, New Mexico
> >
> >
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:37:23 -0600
> From: "Bennett, Catherine (Katie)" <cbennett@lrri.org>
> Subject: RE: Antigen Retrieval, Vimentin & BrdU
>
> One idea that has been posed to me is to do the Vimentin stain first.
Then
> to do the microwave AR, protease, and HCl pre-treatments then the BrdU
> staining.  Might I loose the Vimentin stain if I go in this order?  (I'm
> using a Vector ABC-AP kit and Vector Red as the chromagin for Vimentin and
a
> Vector ABC kit with DAB for the BrdU.)
>
>
>
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:37:52 -0600
> From: "Tarpley, John" <jtarpley@amgen.com>
> Subject: RE: Antigen Retrieval
>
> Katie,
> You don't mention which antibody you do first, but here's a suggestion
that
> works for us. First, do the vimentin since it doesn't require any
> pretreatment for your method. I'm assuming here that you're doing one
> antibody with HRP and the second with Alk Phos. If not, you'll have to
> adjust accordingly. Following this assumption you would detect the
vimentin
> using HRP with DAB. Following your wash steps after DAB do the antigen
> retrieval. We use Citrate Buffer and DAB is not harmed by the retrieval,
> enzyme, or acid step. Now do the BrdU stain and detect with Alk Phos. If
you
> want permanent sections you could use Vector Red as the chromagen. It has
> the added advantage that it is fluorescent. Best of luck.
>
> John Tarpley 15-2-B
> Associate Scientist
> Specialist Image Analysis & Immunohistochemistry
> Amgen Inc
> One Amgen Center Drive
> Thousand Oaks, CA  91320
>
>
> > ----------
> > From: Bennett, Catherine (Katie)[SMTP:cbennett@lrri.org]
> > Sent: Thursday, December 23, 1999 8:51 AM
> > To: 'Histonet Server'
> > Subject: Antigen Retrieval
> >
> > I realize not many histonetters may be online right now, but to those
out
> > there, I present my current dilemma.
> >
> > I am trying to do double immuno staining for BrdU and Vimentin in 10%
> > neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> > labeling with almost all of the microwave antigen retrieval procedures
> > I've
> > been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> > EDTA).  But the Vimentin stain after microwave AR is really weak.
Without
> > antigen retrieval, I get no BrdU staining but awesome Vimentin stains.
My
> > protocol always includes a 5 minute 0.05% Protease K enzyme digestion
> > step,
> > and a 1h HCL denaturation step.  Without the microwave AR, these alone
> > won't
> > do the trick for the BrdU.
> >
> > I'm guessing my next step is to ditch the microwave AR since it kills
the
> > vimentin and play around with various enzyme digestion times to boost
the
> > BrdU.
> >
> > Anyone have any insight or suggestions?
> >
> >
> >
> > ****************************
> > Catherine "Katie" Bresee Bennett
> > Sr. Technical Associate
> > Lovelace Respiratory Research Institute
> > Albuquerque, New Mexico
> >
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:38:51 -0600
> From: "Kopczynski, Charlotte" <Charlotte.Kopczynski@baycare.org>
> Subject: RE: Handling Sentinel Lymph Node Biopsies
>
> We have been doing these cases for over a year now.  During the first
year,
> the specimens were sent from surgery to nuclear medicine and held for 60
> hours before being sent to Histology. Nuclear Medicine monitored all year
> and then it was decided by our physicist and the medical staff that it was
> safe for the specimens to come directly to Histology.
>
> Charlotte Kopczynski
> Supervisor Pathology/Histology
> Morton Plant Hospital
> Clearwater, Florida 34683
>
>
> > -----Original Message-----
> > From: Decarli, Terri [SMTP:TerDec@northarundel.org]
> > Sent: Wednesday, December 22, 1999 5:04 PM
> > To: 'Histonet@pathology.swmed.edu'
> > Cc: Decarli, Terri
> > Subject: Handling Sentinel Lymph Node Biopsies
> >
> > We are in the research stage of performing these isotope injected
> > specimens
> > along with the blue dye.  I would like to know how you are handling
these
> > specimens, are any personnel being monitored for the so-called low
dosage
> > of
> > radiation,  how long from time of surgery to grossing in Pathology. Any
> > information you can give me will be helpful.  Thank you
> >
> > My E mail address is:
> >
> > TerDec@North <mailto:TerDec@North> Arundel.org
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 12:39:16 -0600
> From: "Kopczynski, Charlotte" <Charlotte.Kopczynski@baycare.org>
> Subject: Happy Holidays
>
> This past year has been quite exciting and I have enjoyed being a part of
> the Histonet.  The experience and knowledge has been astounding.  I hope
you
> all have wonderful holidays and that the New Year brings happiness.
>
> Charlotte Kopczynski
> Supervisor Pathology/Histology
> Morton Plant Hospital
> Clearwater, Florida 34683
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 13:46:00 -0600
> From: "Joyce Kotzuk" <JKotzuk@salud.unm.edu>
> Subject: Re: Antigen Retrieval
>
> Can you do the vimentin first, then do HIER and the BrdU staining second?
> I don't know if it would work, if the HIER would somehow ruin the vimentin
> staining, but it may be worth a try.
> Joyce Kotzuk, Univ. of New Mexico pathology dept.
>
> >>> "Bennett, Catherine (Katie)" <cbennett@lrri.org> 12/23/99 09:51AM >>>
> I realize not many histonetters may be online right now, but to those out
> there, I present my current dilemma.
>
> I am trying to do double immuno staining for BrdU and Vimentin in 10%
> neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> labeling with almost all of the microwave antigen retrieval procedures
I've
> been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> EDTA).  But the Vimentin stain after microwave AR is really weak.  Without
> antigen retrieval, I get no BrdU staining but awesome Vimentin stains.  My
> protocol always includes a 5 minute 0.05% Protease K enzyme digestion
step,
> and a 1h HCL denaturation step.  Without the microwave AR, these alone
won't
> do the trick for the BrdU.
>
> I'm guessing my next step is to ditch the microwave AR since it kills the
> vimentin and play around with various enzyme digestion times to boost the
> BrdU.
>
> Anyone have any insight or suggestions?
>
>
>
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 13:46:36 -0600
> From: Cynthia Favara <cfavara@niaid.nih.gov>
> Subject: RE: Antigen Retrieval
>
> Katie,
> I have done double staining for F480/gp70 with DAB as the substrate
> and then followed with a second stain after HIER.  This is is a paper to
be
> released in The Journal of Virology Jan 2000 74:465-473. We had the same
> problem as F480 was destroyed by HIER but we needed it for the gp70. I
> believe the paper is available over the net or i would be happy to send
you
> a copy.
> Cynthia Favara
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 13:47:07 -0600
> From: Cynthia Favara <cfavara@niaid.nih.gov>
> Subject: RE: Cells
>
> How about histogel???
> http://www.labstore.com/histogel.html
> Or you could grow cells on coverslips.....
> Cynthia Favara
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 13:47:54 -0600
> From: "Weems, Joyce" <JWEEMS@sjha.org>
> Subject: RE: Cells
>
> How about the Histogel from Richard Allen? It works well for our cell
> blocks!
> Joyce Weems
> Pathology Manager
> Saint Joseph's Hospital of Atlanta
>
>
> -----Original Message-----
> From: Lynn Gardner [SMTP:lynn-gardner@uiowa.edu]
> Sent: Thursday, December 23, 1999 12:28 PM
> To: Histonet@pathology.swmed.edu
> Subject: Cells
>
> Dear Histonetters,
>
> First of all Merry Christmas and Happy New Year to all!! I have a
> question
> that hopefully someone will have an answer.
>
> We have grown up some cells in a petri dish. We are now wanting to
> try and
> process these cells, however, we need to keep them in their shape
> and not
> disrupt them. Does anyone have an idea of how to do this or
> reference to
> journal articles that would contain this information?
>
> I thought we could place the cells in agar and then process them but
> don't
> know what type of agar would process and cut well. Would like any
> imput
> possible.
>
> Also, is there anyone out there that knows of a cytology network
> like our
> histonet?
>
> Thanks for your help everyone! Happy Holidays!
>
> Sincerely,
> Lynn Gardner
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 14:56:40 -0600
> From: tom@adpath.com
> Subject: Re: Antigen Retrieval, Vimentin & BrdU
>
> Hi Katie:  I would think the trick is in which one of the substrates
> will survive the MW/AR step.  DAB  would be more likely to survive than
> the vector red altho you can try it and see.  Since BRDU is in the
> nuclei and the vimentin out in the cytoplasm, you could try two colors
> of DAB also.  Tom
>
> "Bennett, Catherine (Katie)" wrote:
> >
> > One idea that has been posed to me is to do the Vimentin stain first.
Then
> > to do the microwave AR, protease, and HCl pre-treatments then the BrdU
> > staining.  Might I loose the Vimentin stain if I go in this order?  (I'm
> > using a Vector ABC-AP kit and Vector Red as the chromagin for Vimentin
and a
> > Vector ABC kit with DAB for the BrdU.)
> >
> > ****************************
> > Catherine "Katie" Bresee Bennett
> > Sr. Technical Associate
> > Lovelace Respiratory Research Institute
> > Albuquerque, New Mexico
>
> - --
> *******************************
> Thomas J. Kuwahara
> Senior Immunohistochemist
> Advanced Pathology Systems
> 3801 Sacramento St. suite 621
> San Francisco, CA 94118
> 415 750 6800 x23067 tel
> 415 750 2332 fax
> tom@adpath.com
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 15:20:40 -0600
> From: Gayle Callis <uvsgc@msu.oscs.montana.edu>
> Subject: sheep antibodies
>
> SeroTec, Kirkegaard Perry, and Jackson Immunoresearch
> All have websites
>
> Gayle Callis
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 15:37:52 -0600
> From: Gayle Callis <uvsgc@msu.oscs.montana.edu>
> Subject: cell plug preps
>
> I learned some of the neatest tricks for handling cells in Barb Wright's
> workshop, substituting for John Tarpley, and it works beautifully.
>
> You can make the cells release or scrape off, whatever is done to get them
> floating, put them in a 50 ml tube, centrifuge, spin down, pour off
> supernate, do this a couple of time more with pure PBS, then add
> OCT to top of plug, snap freeze the bottom, pop plug out, double embed it
> in OCT.  Or better yet, take the cells and make a suspension, purchase
> CollaPlugs (sorry, I'm at home and not near company info) which are
collagen
> plugs used by dentists for wound healing, the resorbable type, soak up the
> cell suspension in this plug.  The plug can then be cut into 3 pieces,
> and each piece fixed with a different fixative then paraffin process, or
> or embed in OCT,  snap freeze for frozen sections.  You can
> then stain the cells, which are maintained without being slammed against
> a slide by a cytospin, or messed up, the cells looked wonderful!
>
> Don't try to use a 15 ml conical, the tip is too narrow. The snap frozen
> plug has a very high number of cells, so cut at 4 um for frozens, and
> the paraffin embedded plug is treated just like a tissue.
>
> Have fun, and boy did this ever work.
>
> Gayle Callis
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 15:57:35 -0600
> From: "ANN MARUSKA" <amarusk1@FAIRVIEW.ORG>
> Subject: Re: ER/PR, Her2 Regulatory Questions
>
> Louri,
> I agree with Amos' reply and do pretty much the same thing in our lab -
being
> able to follow the instructions to a "T" is a must.  However, we mount a
> patient test tissue on the same slide as a control tissue (one from our
lab)
> in addition to the validation.  Just an extra step to make sure everything
has
> worked fine and the slide layout was correct.
> As a side note, we just learned about a month or two ago that the
validation
> slides should also be refrigerated.  Up until then we had been holding
them at
> room temp. and using one for the daily run.
> Ann Maruska
> Fairview-University Med Ctr
> Mpls. MN
> amarusk1@fairview.org
>
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 16:14:24 -0600
> From: "Tarpley, John" <jtarpley@amgen.com>
> Subject: RE: cell plug preps
>
>
>
> > ----------
> > From: Gayle Callis[SMTP:uvsgc@msu.oscs.montana.edu]
> > Sent: Thursday, December 23, 1999 1:17 PM
> > To: histonet@pathology.swmed.edu
> > Subject: cell plug preps
> >
> --snip snip--
>   Or better yet, take the cells and make a suspension, purchase
> > CollaPlugs (sorry, I'm at home and not near company info) which are
> > collagen
> > plugs used by dentists for wound healing, the resorbable type, soak up
the
> > cell suspension in this plug.  The plug can then be cut into 3 pieces,
> > and each piece fixed with a different fixative then paraffin process, or
> > or embed in OCT,  snap freeze for frozen sections.  You can
> > then stain the cells, which are maintained without being slammed against
> > a slide by a cytospin, or messed up, the cells looked wonderful!
> CollaPlugs are by Calcitek cat#0102. 2320 Faraday Avenue, Carlsbad, CA
92008
> 800 854 7019. I haven't bought any for awhile, but the last price I have
is
> $63/box of 10 which equals 30 cell preps. The only problem with using the
> plugs as Gayle describes might be that in the original message the point
was
> made that the original morphology of the cells needs to be maintained.
That
> won't happen if you trypsinize the cells off the plates and scraping may
> disrupt at least some of the cells so that may be a problem. Some cell
> lines, not all, will actually grow into the plug so that's really the best
> option if you can setup new cultures.
>
> - --snip snip--
>
>
>
> > Gayle Callis
> >
>
>
> ----------------------------------------------------------------------
>
> Date: 23 Dec 1999 17:15:17 -0600
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Tris-EDTA
>
> On Tue, 21 Dec 1999, lcjx@pdq.net wrote:
>
> > I'm looking for a Tris-EDTA recipe for heat induced antigen retrieval.
> Thanks
> > in advance and happy holidays!
>
> Here are 2 references. I haven't tried either method myself.
>
>   Balaton,AJ et 4 al. 1995. Protocole "EDTA-autocuiseur." Une technique
>   immunohistochimique performante Annales de Pathologie 15, 295. (Letter)
>     Rehydrate formalin-fixed paraffin sections. Put in 2 litres of boiled
>   inM EDTA (adjusted to pH 8) in pressure cooker. Lid on, and leave 1.5
>   min when at full pressure. Cool under tap. More convenient than
>   microwave oven.
>
>   Pileri, SA et 15 al. 1997. Antigen retrieval techniques in
>   immunohistochemistry: Comparison of different methods
>   Journal of Pathology 183, 116-123.
>     61 antibodies tested. 1 mM EDTA-NaOH (pH 8) was better generally
>   than citrate (6.0) or TRIS buffer (8.0) or protease XIV (5 m, 37C).
>   Most convenient treatment was in pressure cooker for 2 min. Some
>   antibodies better with the other methods.
>
>   Both methods appear to be very similar. The second paper is a
>   particularly thorough study.
>
>   Good luck retrieving, and have a good Christmas,
>
>  John A. Kiernan,
>  Department of Anatomy & Cell Biology,
>  The University of Western Ontario,
>  LONDON,  Canada  N6A 5C1
>    E-mail: kiernan@uwo.ca
>
>
>
>
> Here are the messages received yesterday!