RE: Daily Digest
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| From: | "Culberson, Cathy" <CCulberson@carolinas.org> (by way of histonet) |
| To: | histonet@histosearch.com |
| Reply-To: | |
| Content-Type: | text/plain; charset="us-ascii" |
Cathy Culberson, MS
Supervisor, General Surgery Research
(704)355-2605
Email:cculberson@carolinas.org
-----Original Message-----
From: HistoNet Server [SMTP:histonet@pathology.swmed.edu]
Sent: Friday, December 24, 1999 1:16 AM
To: HistoNet Server
Subject: Daily Digest
----------------------------------------------------------------------
Date: 23 Dec 1999 04:45:23 -0600
From: "Sabrina Dize" <Dize_Sabrina@piedmont.promina.org
<mailto: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 <mailto:ldize@mindspring.com>
Dize_Sabrina@piedmont.promina.org
<mailto: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
<mailto: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
<mailto: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 <mailto: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 <mailto:schiller@instrumedics.com>
----------------------------------------------------------------------
Date: 23 Dec 1999 10:45:12 -0600
From: thaxtonpm@aol.com <mailto: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:
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Your card number is: 2308314150176
(enter this number in the space provided under "Pickup Card" in the
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Alternatively you can pick it up by clicking on the link below (For
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http://www.webmaniadesigns.com/cgi-bin/magiccard.cgi?2308314150176
<http://www.webmaniadesigns.com/cgi-bin/magiccard.cgi?2308314150176>
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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
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<http://freewebcards.com/save.shtml> . We recommend that you save your card
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If you are having problems retrieving your card, please go to
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If you are having problems going to my help page, please send an
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<mailto: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
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----------------------------------------------------------------------
Date: 23 Dec 1999 11:01:17 -0600
From: "Bennett, Catherine (Katie)" <cbennett@lrri.org
<mailto: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 <mailto: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
<http://www.hotmail.com>
----------------------------------------------------------------------
Date: 23 Dec 1999 11:45:53 -0600
From: Lynn Gardner <lynn-gardner@uiowa.edu
<mailto: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 <mailto: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 <mailto: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
<mailto:cbennett@lrri.org> >
To: 'Histonet Server' <histonet@pathology.swmed.edu
<mailto: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
<mailto: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
<mailto: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]
<mailto:[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
<mailto: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]
<mailto:[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> <mailto:TerDec@North
<mailto:TerDec@North> > Arundel.org
----------------------------------------------------------------------
Date: 23 Dec 1999 12:39:16 -0600
From: "Kopczynski, Charlotte" <Charlotte.Kopczynski@baycare.org
<mailto: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
<mailto: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
<mailto: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
<mailto: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
<mailto:cfavara@niaid.nih.gov> >
Subject: RE: Cells
How about histogel???
http://www.labstore.com/histogel.html
<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 <mailto: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]
<mailto:[SMTP:lynn-gardner@uiowa.edu]>
Sent: Thursday, December 23, 1999 12:28 PM
To: Histonet@pathology.swmed.edu
<mailto: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 <mailto: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 <mailto:tom@adpath.com>
----------------------------------------------------------------------
Date: 23 Dec 1999 15:20:40 -0600
From: Gayle Callis <uvsgc@msu.oscs.montana.edu
<mailto: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
<mailto: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
<mailto: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 <mailto:amarusk1@fairview.org>
----------------------------------------------------------------------
Date: 23 Dec 1999 16:14:24 -0600
From: "Tarpley, John" <jtarpley@amgen.com
<mailto:jtarpley@amgen.com> >
Subject: RE: cell plug preps
> ----------
> From: Gayle Callis[SMTP:uvsgc@msu.oscs.montana.edu]
<mailto:[SMTP:uvsgc@msu.oscs.montana.edu]>
> Sent: Thursday, December 23, 1999 1:17 PM
> To: histonet@pathology.swmed.edu
<mailto: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
<mailto:jkiernan@julian.uwo.ca> >
Subject: Re: Tris-EDTA
On Tue, 21 Dec 1999, lcjx@pdq.net <mailto: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 <mailto:kiernan@uwo.ca>
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