[Histonet] Problem with section frozen human eye lens
Madary, Joseph
MadaryJ <@t> MedImmune.com
Mon Mar 15 08:46:11 CDT 2010
Any netters have some idea about sectioning human eye without losing the lens part way through? A softening treatment that will allow for frozen sectioning, a special temperature?
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
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Subject: Histonet Digest, Vol 76, Issue 20
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Today's Topics:
1. IHC on 3 year fixed tissue? (Kevin Egan)
2. Histonet (baumannr <@t> med.uni-marburg.de)
3. Re: IHC on 3 year fixed tissue? (Rene J Buesa)
4. RE: IHC on 3 year fixed tissue? (Jacqui Detmar)
5. in situ hybridization histochemistry question about
radioisotope (Susan Bachus)
6. Hello everyone, I am new to this forum (Valerie Rodriguez)
7. Question about new Frieda Carson book (Valerie Rodriguez)
8. Histonet (Valerie Rodriguez)
9. Question about new Frieda Carson book (Valerie Rodriguez)
----------------------------------------------------------------------
Message: 1
Date: Sat, 13 Mar 2010 14:15:12 -0500
From: Kevin Egan <kevinpe <@t> mail.med.upenn.edu>
Subject: [Histonet] IHC on 3 year fixed tissue?
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <4B9BE440.1020803 <@t> upenn.edu>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed
Hello Histonetters,
I've been asked to perform anti-GFP IHC on some mouse bones which have
been sitting in NBF for 3-4 years. My Abcam rabbit polyclonal gets great
results in GFP mouse control tissue, but I can't seem to get any results
out of these bones. I've tried citrate buffer HIER using autoclave,
hotplate, and 65 degree water batch over night, but my signal is
non-existent (or really the noise is so huge you can't distinguish).Do
the great minds of histonet have any ideas on how to achieve good
staining? Is enzymatic epitope retrieval worth a try? Does anyone have
a time machine I can use to stop my predecessors from just sticking the
bones in NBF and forgetting about them for years?
This listserv has helped me countless times, I am truly appreciative of
the knowledge and experience available on Histonet.
Thanks,
Kevin
------------------------------
Message: 2
Date: Sat, 13 Mar 2010 21:41:58 +0100
From: baumannr <@t> med.uni-marburg.de
Subject: [Histonet] Histonet
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<20100313214158.xyuwbumfk84g0s8k <@t> webmail.med.uni-marburg.de>
Content-Type: text/plain; charset=ISO-8859-1; DelSp="Yes";
format="flowed"
dear Val, first my english is not good. I can understand....but the grammar!
My way is similar- after studies I wanted to work in microbiologie.
But I´ve got a job in Pathologie. First time I was alone in the
laboratorie.I had to manage the routine...cutting,staining,preparation
and so on...and that was at the beginning after my studies, I had no
experiances! Sometimes I thought " OH GOD " but I think, this is the
best way to learn! I have made my mistakes! -but I´ve learned.I´ve
never made the same mistakes two times! Now I do my job about 36 years
and I teach students in histotechnologie.
Val, I think there is no problem to make a mistake at the beginning!
For your " beginning " I wish you good luck.
Best wishes Renate
------------------------------
Message: 3
Date: Sat, 13 Mar 2010 15:17:49 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] IHC on 3 year fixed tissue?
To: histonet <@t> lists.utsouthwestern.edu, Kevin Egan
<kevinpe <@t> mail.med.upenn.edu>
Message-ID: <706114.82676.qm <@t> web65710.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
I have to imagine that you are doing the HIER in the FFPE sections? If you have used autoclave unsuccessfully it seems that the epitopes are so cross-linked that they do not respond to the treatment.
I would try a piece of fixed tissue before processing, cut it in a very thin slice (about 2 mm maximum) and heat it in distilled water at 65ºC for 30 minutes. Wah it thoroughly and process it as usual.
After that, prepare the sections and HIER them in citrate buffer in a steamer and proceed as usual.
By doing this you will have to steps were the cross-linkages area going to be weaken and perhaps you will get some acceptable reaction.
René J.
--- On Sat, 3/13/10, Kevin Egan <kevinpe <@t> mail.med.upenn.edu> wrote:
From: Kevin Egan <kevinpe <@t> mail.med.upenn.edu>
Subject: [Histonet] IHC on 3 year fixed tissue?
To: histonet <@t> lists.utsouthwestern.edu
Date: Saturday, March 13, 2010, 2:15 PM
Hello Histonetters,
I've been asked to perform anti-GFP IHC on some mouse bones which have been sitting in NBF for 3-4 years. My Abcam rabbit polyclonal gets great results in GFP mouse control tissue, but I can't seem to get any results out of these bones. I've tried citrate buffer HIER using autoclave, hotplate, and 65 degree water batch over night, but my signal is non-existent (or really the noise is so huge you can't distinguish).Do the great minds of histonet have any ideas on how to achieve good staining? Is enzymatic epitope retrieval worth a try? Does anyone have a time machine I can use to stop my predecessors from just sticking the bones in NBF and forgetting about them for years?
This listserv has helped me countless times, I am truly appreciative of the knowledge and experience available on Histonet.
Thanks,
Kevin
_______________________________________________
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Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 4
Date: Sat, 13 Mar 2010 22:18:17 -0500
From: "Jacqui Detmar" <detmar <@t> lunenfeld.ca>
Subject: RE: [Histonet] IHC on 3 year fixed tissue?
To: "Rene J Buesa" <rjbuesa <@t> yahoo.com>,
<histonet <@t> lists.utsouthwestern.edu>, "Kevin Egan"
<kevinpe <@t> mail.med.upenn.edu>
Message-ID:
<A249C197854D3442BDAE4C6BA4D5553C01039238 <@t> ex1.ad.mshri.on.ca>
Content-Type: text/plain; charset="iso-8859-1"
Hey there. I have done IHC on mouse placentae that have been fixed in NBF for over 2 years and it took me 5-10 minutes of ProK (10 ug/ml in PBS) followed by 60 minutes of HIER in 10 mM citrate buffer (made sure PBS was at pH 6) in a veggie steamer.
I had previously done this IHC in a veggie steamer for only 25 minutes in 10 mM citrate buffer (pH 6) for mouse placentae that had only been fixed for 24 hours in NBF. The antigen was a nuclear receptor (AhR); I have found nuclear antigens to be slightly more difficult to retrieve...at least for mouse placenta! Note that the 2-year-fixed tissue did NOT react positively until the 60-minute mark after HIER, although I tested at 25, 35 and 50 minutes. Only 60 minutes HIER plus enzyme retrieval worked, even though "quick-fixed" tissue worked simply with 25 minutes HIER. Definitely worth a try, if you're desperate. It took me over a year to get this **#&%*#&%** tissue working! <grin> Also, used a 1:100 dilution instead of my normal 1:500 dilution of the antibody, so you might want to titrate the antibody, too.
Am I being lucid? Have just come home from a most excellent dinner party! Perhaps not such a good idea to give histo-advice on a Saturday night....but I deeply sympathized with your plight <grin>. If you have any questions, please feel free to contact me personally, Kevin, either on gmail or at my SLRI account.
Jacqui
Jacqui Detmar
Work phone: 1-416-646-0223
Work fax: 1-416-862-9696
Cell phone: 1-647-273-8735
jacquidetmar <@t> gmail.com
________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Rene J Buesa
Sent: Sat 3/13/2010 6:17 PM
To: histonet <@t> lists.utsouthwestern.edu; Kevin Egan
Subject: Re: [Histonet] IHC on 3 year fixed tissue?
I have to imagine that you are doing the HIER in the FFPE sections? If you have used autoclave unsuccessfully it seems that the epitopes are so cross-linked that they do not respond to the treatment.
I would try a piece of fixed tissue before processing, cut it in a very thin slice (about 2 mm maximum) and heat it in distilled water at 65ºC for 30 minutes. Wah it thoroughly and process it as usual.
After that, prepare the sections and HIER them in citrate buffer in a steamer and proceed as usual.
By doing this you will have to steps were the cross-linkages area going to be weaken and perhaps you will get some acceptable reaction.
René J.
--- On Sat, 3/13/10, Kevin Egan <kevinpe <@t> mail.med.upenn.edu> wrote:
From: Kevin Egan <kevinpe <@t> mail.med.upenn.edu>
Subject: [Histonet] IHC on 3 year fixed tissue?
To: histonet <@t> lists.utsouthwestern.edu
Date: Saturday, March 13, 2010, 2:15 PM
Hello Histonetters,
I've been asked to perform anti-GFP IHC on some mouse bones which have been sitting in NBF for 3-4 years. My Abcam rabbit polyclonal gets great results in GFP mouse control tissue, but I can't seem to get any results out of these bones. I've tried citrate buffer HIER using autoclave, hotplate, and 65 degree water batch over night, but my signal is non-existent (or really the noise is so huge you can't distinguish).Do the great minds of histonet have any ideas on how to achieve good staining? Is enzymatic epitope retrieval worth a try? Does anyone have a time machine I can use to stop my predecessors from just sticking the bones in NBF and forgetting about them for years?
This listserv has helped me countless times, I am truly appreciative of the knowledge and experience available on Histonet.
Thanks,
Kevin
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 5
Date: Sun, 14 Mar 2010 02:28:12 -0500
From: "Susan Bachus" <susanbachus <@t> verizon.net>
Subject: [Histonet] in situ hybridization histochemistry question
about radioisotope
To: "histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <58C8F3C807724CEA82DB1846B70D1928 <@t> OwnerPC>
Content-Type: text/plain; charset="iso-8859-1"
I have routinely seen that we obtain slightly different results (like 10-20%
variance) in using 35S dATP for TdT-catalyzed tailing reactions of
oligonucleotides from month to month as we use different batches (made fresh
monthly) of the 35S dATP. But on a few occasions over the years we have
found dramatically reduced results with a particular lot. This happened
again in February, and I confirmed by comparing directly between the Feb. &
March lots in March that the results with the Feb. lot were half that of the
March batch (of course, given the half-life of 35S, they would be expected
to be down some, but not by half!). The company was good about believing me
and not charging us for the March "replacement" batch, but they have no idea
what could cause this to happen occasionally. They check the specific
activity, pH, purity, etc. and run a "bio-assay" that is a binding assay
each month, but were not set up to test the enzymatic reaction. I told
them that years ago when this happened it was finally determined that the
DTT concentration was unusually high and interfered with the reaction, but
they discounted this possibility. I guess I should just count my blessings
that things are working again now (mercifully, I was able to cling to
optimism, based on past experience, that the isotope was the problem, until
I could confirm this myself), but it's very frustrating that the company is
not motivated to check the efficacy of the item for this particular
application (which I would imagine a lot of customers use it for) each
month, or to figure out what causes this to happen on occasion (so as to
avoid it!)--we lose a lot of time and other expensive reagents (e.g. the
TdT) each time this happens. I also don't know how to interpret it--I know
that as the enzyme begins to degrade it just means that the tails are
shorter, which I can compensate for with a longer film exposure, but I have
no idea what's going on when the isotope causes the problem & am afraid to
use those batches. Does anyone else using ISH have any ideas about what
causes this variability? grateful for any thoughts, Susan
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Message: 6
Date: Sun, 14 Mar 2010 05:45:51 -0700
From: Valerie Rodriguez <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] Hello everyone, I am new to this forum
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<b9208e7b1003140545k133b4297qad654959cb669142 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
Val, the life is very funny.
I graduated in Chemical Engineering at the University of Pisa. I'm Italian.
I worked in a mechanical company: Fiat.
Since I was 15teen my greater hobby was the microscope. Now I am a pensioner
and my hobby goes on with Histology.
On FaceBook (FB) I have a lot of histologist friends.
If you join me on FB (massimo.tosi_m <@t> libero.it) I think they could help you.
Thank you.
With my Best Regards,
Massimo Tosi
Yes, life is funny, considering that I first wanted to be a cytotech, but I
could not, so I ended up being an histotech and ended up liking histology
way more, and now I am working in a lab that focus more in cytopathology.
There is no microtomes, embedding machines, just a cryostat, and frozen
section stains and the cytology stains. I am not doing the smears yet,
another tech is doing it, but soon they will train me how to assist the
pathologist in a fine needle aspiration just in case the other tech can't do
it. I just stain the PAP slides, coverslip them, organize them etc, but
still it is good to know cytology so that when something wrong happens, I
can troubleshoot the problem.
The people from the lab assumes that histology and cytology are very alike
but they are not. One of them told me that you stains slides the same, and
that got me dumbfounded because in histology you don't use H20 saline as a
dehydrant solution. If I were a cytotech I would knew already that I had to
change the H20 saline frequently because is the solution that gets more
dirty after staining a few racks of slides.
In cytology hematoxylin is used, but orange,and EA, are not used in
histology. The bluing solution is almost the same, it used to be lithium
carbonate in the lab but seems they changed it to another chemical. They use
8 containers of alcohol 95%, and 3 of 99% when I compared that to the H&e
protocol of my histology textbook cytology uses a looooot more alcohol, so
the solutions are not the same. Plus you must be very careful with the
slides because they don't have more cytology samples, unlike histology if a
slide breaks you can look for that block of tissue and you cut a new slide,
that is not possible in cytology, well in their lab, because I ask this and
they told me that if I mess up a slide, then the pathologist has to obtain a
new sample from the patient, so I think cytology is a little more difficult
than histology for this reason.
Seems that these two careers are slowly getting incorporated. In my country
seems that this happen long time ago, but in the U.S I think cytology and
histology is going to become one profession. The new Frieda Carson
Histotechnology textbook has a new chapter about cytoprepatation. This book
appeared in amazon stores online like a month after I graduated histotech
school, so too bad it did not came before because I could have bough it
instead of the old version I have. I will ask a question about this book in
the forum later.
We'll thanks for the reply.
------------------------------
Message: 7
Date: Sun, 14 Mar 2010 06:00:32 -0700
From: Valerie Rodriguez <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] Question about new Frieda Carson book
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<b9208e7b1003140600t17307616x2032c5a1db0fe0ea <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
I have a question about the new Frieda Carson book: Histotechnology: A
Self-Instructional Text 3rd edition. In this book there is a new chapter
about cytopreparation techniques. I will like to know if this chapter
teaches you how to troubleshoot problems in the staining of the slides, how
to do fine needle aspirations and other techniques in cytology, or it just
gives you a brief review of how to prepare cytology?????. I have the
previous version, the new book came right after I graduate histotech school.
Is it worth to buy the new book? Someone told me the pictures and text of
all the other chapters have not changed, just the new cytology chapter.
My lab is concentrated more in fine needle aspirations (cytology) than
histology so I think it will be good for me to buy the new edition since is
like a chapter made just for histotech so that they can undersand cytology
better, but since is new is a little pricey. I may wait until the price is a
little lower. Frieda's book is very excelent in preparing histotechs on how
to troubleshoot problems, but I will like to find a book like that but for
cytotechnology.
What are your opinions????
------------------------------
Message: 8
Date: Sun, 14 Mar 2010 09:14:28 -0700
From: Valerie Rodriguez <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] Histonet
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<b9208e7b1003140914g220e5c69v6d5aca9129cf545d <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
Thank You very much.English is not my first language also. My native
language is Spanish, so I also make grammar mistakes once in a while :). I
meet an histotechnician who first got a bachelor degree in microbiology but
could not get a job, and he started to volunteer in a pathology lab. All he
did was simple tasks just as labeling and filing slides, then he got trained
in histology since he showed interest. Since he has no degree in histotech
only the HT licence of PR he was not payed very well so he decided to pursue
his studies in cytotechnology, now I think he will graduate this year during
the summer. Sometimes we prepared ourselves for a specialization and ended
up something entirely different. That's how life is, but as you say the best
thing is to prepare yourself for everything to say competitive in the
clinical laboratory science field. There are labs in Puerto Rico where even
the histotech has to work in the reception area!.
Your story is inspiring, this is my first time working, so I have a lof of
pressure, since I think I am the only HTL in Puerto Rico who got a histotech
degree in the U.S, the rest here are just trained. Very few also have the
ASCP certification, the government of puerto rico provide their own
licenses. A university in Puerto Rico used to offer a 1 program course of
histotechnology but I think they stopped the program in the early 90's.
Since I got educated in the U.S the employees in the lab expect me to know
everything. I have no work experience, but I had my internship in the U.S,
then I worked as a volunteer in two diffent pathology laboratories in Puerto
Rico, the way they organize their system is very different, so in my first
two weeks it has taken time to understand their system. I started off good
but I have made my mistakes but from your mistakes you learn. I hope I keep
pursuing this career for a long time like you and have the chance to teach
students in a histotech school. I wish I could open one here, it is very
important.
We'll take care.
------------------------------
Message: 9
Date: Sun, 14 Mar 2010 09:23:53 -0700
From: Valerie Rodriguez <vrodriguez10 <@t> gmail.com>
Subject: [Histonet] Question about new Frieda Carson book
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<b9208e7b1003140923m706921a6q3710a88314ae184c <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
No, I don't have the book, for how much are you selling it? I am looking for
a very economic book, not too expensive, since cytotech is not really my
profession, I just want a guide just to have some knowledge of cytotech. I
am also interested in the new edition of frieda textbook but if your book is
more complete then it may be better. I don't need to know about diagnostic
cytopathology just the technical part.
Thanks
------------------------------
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