Re: [Histonet] Fixed frozen non-paraffin mouse brain

[John Kiernan via Histonet]

Dear Ed,

Adequate fixation is important. Formaldehyde penetrates quickly but reacts 
slowly with proteins. 4% formaldehyde made by depolymerizing paraformaldehyde 
(an insoluble high polymer) is the same as formaldehyde made by 10X dilution of 
formalin (a mixture of soluble low polymers).

For cryoprotection the sucrose must thoroughly penetrate the fixed specimen, 
which must sink in the concentrated solution.

Your Swiss cheese artifact is due to slow freezing. In badly frozen brains the 
ice crystal holes are as big as large neurons and the tissue architecture is 
destroyed.

Unless specimens are really tiny and frozen super-fast (special methods for 
electron microscopy), ice crystals always form and show later as holes in the 
sections. You get bigger holes with bigger specimens and slower freezing.
With very fast freezing, a skilled technician can get good cryostat sections 
even of small unfixed specimens such as muscle biopsies. These are needed for 
enzyme activity histochemistry methods used in diagnostic pathology and in 
research.

Cryoprotection of fixed specimens slows the growth of ice crystals. With luck, 
the holes are too small to interfere with light microscope studies of sections. 
In neuroscience research, quite thick frozen sections of samll animals' brains 
have been the norm for more than 50 years.

About 20 years ago I wrote a chapter that gave some quite detailed instructions 
and explanations, with references. (Don't do anything important just because a 
chapter or a review says so; check at least some of the refs!)   Here is the 
reference.

Kiernan, J. A. 2002. Freezing and fixation. Chapter 8 in Microscopy and 
Histology for Molecular Biologists. A User's Guide, ed. Kiernan, J. A. & Mason, 
I. G. pp. 103-143.  London: Portland Press.  ISBN 1855781417.

Your university's library in Urbana might have the book. It's out-of-print with 
its publisher. There are used copies on the web for much less than the original 
price.

John Kiernan
Emeritus neuroanatomist and histochemist
London, Canada
https://www.schulich.uwo.ca/anatomy/people/bios/emeriti/kiernan_john.html
= = =

From: Roy, Edward J via Histonet 
Sent: 04 July 2020 20:08
To: histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Fixed frozen non-paraffin mouse brain

As a research lab, we sometimes would like to use paraformaldehyde-fixed but 
non-paraffin embedded tissues; paraffin embedding alters antigens and 
necessitates antigen retrieval, but simple fixation does not. We have done the 
traditional 30% sucrose before OCT and freezing, with cryostat sectioning, but 
results are inconsistent, sometimes producing Swiss-cheese brains. Does anybody 
have an alternative to 30% sucrose that is more reliable?  I didn’t see 
anything in the Archives after a search for “30% sucrose”.
Thanks very much,
Ed Roy

Edward J. Roy, PhD
Professor Emeritus
Department of Molecular and Integrative Physiology
University of Illinois at Urbana-Champaign
Urbana, IL 61801
217 333-3375


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Re: [Histonet] Fixed frozen non-paraffin mouse brain

[Tina Van Meter via Histonet]

Hi Ed,
I have been using the 30% sucrose technique to cryoprotect animal tissue
for over 40 years without any problem. Did the tissue sink to the bottom of
the specimens jar? After sinking, I blot the excess sucrose from the tissue
on a paper towel before transfering to OCT. What is your procedure to
freeze the tissue?

Thank you,
Tina Van Meter
Scripps Research
Histology Core Manager
Jupiter, FL

On Sat, Jul 4, 2020, 9:26 PM Roy, Edward J via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> As a research lab, we sometimes would like to use paraformaldehyde-fixed
> but non-paraffin embedded tissues; paraffin embedding alters antigens and
> necessitates antigen retrieval, but simple fixation does not. We have done
> the traditional 30% sucrose before OCT and freezing, with cryostat
> sectioning, but results are inconsistent, sometimes producing Swiss-cheese
> brains. Does anybody have an alternative to 30% sucrose that is more
> reliable?  I didn’t see anything in the Archives after a search for “30%
> sucrose”.
> Thanks very much,
> Ed Roy
>
>
> Edward J. Roy, PhD
> Professor Emeritus
> Department of Molecular and Integrative Physiology
> University of Illinois at Urbana-Champaign
> Urbana, IL 61801
> 217 333-3375
>
>
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Re: [Histonet] Tissue processor errors, failures and what to do

[E. Wayne Johnson via Histonet]

Automation is a wonderful thing but it is only a replacement for what people 
used to do by hand.
We have incubators that can be set to 60C and we have a Rube Goldberg-ized 
microwave oven with
a thermal controller and relays (and the not-to-be-forgotten flyback diode) and
a K-type thermal probe covered with aluminum foil (dont try this at home, kids)
so we are ready for any incident or mishap with our cantankerous VIP5.

There is no such thing as a convenient time for it to quit but lots of times 
the trouble
is that it was time to change the solutions and nobody did it even though
anybody could have done it.

If there is nothing we can do to get the VIP5 going again pronto
we finish the processing by hand.
there are repair manuals for some of those machines available online
or you can call the repairman.  If you are out in Bufooee somewhere the repair
manual and the secret repair modes on the machine could be the difference 
between joy
and the slough of despond.

E. Wayne Johnson DVM
Enable AgTech
Beijing

Patpxs via Histonet wrote:

Hi Garrey,

The answer is “it depends”.   What you do when a processor fails depends on the 
failure point.  If the tissue is still in dehydrant it gets treated differently 
than if it fails in the intermediate solvent.

Paula

Sent from my iPhone


On Jul 4, 2020, at 10:08 AM, Garrey Faller via Histonet 
 wrote:

Happy 4th to all.
Does anyone have a procedure on what to do when a tissue processor fails or 
alarms.  I want to learn more about the science behind tissue processing so I 
know what to do when the machine fails. This happened to a friend recently and 
I want to prevent my tissues/biopsies from being ruined.
Thanks.
Garrey

Sent from my iPhone
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Re: [Histonet] Fixed frozen non-paraffin mouse brain

[Porter, Amy via Histonet]

You need to add sucrose to your PFA we use 4%PFA+4%Sucrose to fix and then 
cryoprotect in 30% sucrose which all need to be prepared in Phosphate buffer 
solution NO saline  if you message me directly I would be happy to share 
our SOP this was a very hard thing to learn not a lot in technical 
references about this process.

Sent from my iPhone

> On Jul 4, 2020, at 9:09 PM, Roy, Edward J via Histonet 
>  wrote:
> 
> As a research lab, we sometimes would like to use paraformaldehyde-fixed but 
> non-paraffin embedded tissues; paraffin embedding alters antigens and 
> necessitates antigen retrieval, but simple fixation does not. We have done 
> the traditional 30% sucrose before OCT and freezing, with cryostat 
> sectioning, but results are inconsistent, sometimes producing Swiss-cheese 
> brains. Does anybody have an alternative to 30% sucrose that is more 
> reliable?  I didn’t see anything in the Archives after a search for “30% 
> sucrose”.
> Thanks very much,
> Ed Roy
> 
> 
> Edward J. Roy, PhD
> Professor Emeritus
> Department of Molecular and Integrative Physiology
> University of Illinois at Urbana-Champaign
> Urbana, IL 61801
> 217 333-3375
> 
> 
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>  
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[Histonet] Fixed frozen non-paraffin mouse brain

[Roy, Edward J via Histonet]

As a research lab, we sometimes would like to use paraformaldehyde-fixed but 
non-paraffin embedded tissues; paraffin embedding alters antigens and 
necessitates antigen retrieval, but simple fixation does not. We have done the 
traditional 30% sucrose before OCT and freezing, with cryostat sectioning, but 
results are inconsistent, sometimes producing Swiss-cheese brains. Does anybody 
have an alternative to 30% sucrose that is more reliable?  I didn’t see 
anything in the Archives after a search for “30% sucrose”.
Thanks very much,
Ed Roy


Edward J. Roy, PhD
Professor Emeritus
Department of Molecular and Integrative Physiology
University of Illinois at Urbana-Champaign
Urbana, IL 61801
217 333-3375


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Re: [Histonet] Tissue processor errors, failures and what to do

[Patpxs via Histonet]

Hi Garrey,

The answer is “it depends”.   What you do when a processor fails depends on the 
failure point.  If the tissue is still in dehydrant it gets treated differently 
than if it fails in the intermediate solvent.  

Paula

Sent from my iPhone

> On Jul 4, 2020, at 10:08 AM, Garrey Faller via Histonet 
>  wrote:
> 
> Happy 4th to all.
> Does anyone have a procedure on what to do when a tissue processor fails or 
> alarms.  I want to learn more about the science behind tissue processing so I 
> know what to do when the machine fails. This happened to a friend recently 
> and I want to prevent my tissues/biopsies from being ruined.
> Thanks.
> Garrey
> 
> Sent from my iPhone
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
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[Histonet] Please, unsubscribe

[Mojtaba Akhtari via Histonet]

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[Histonet] Tissue processor errors, failures and what to do

[Garrey Faller via Histonet]

Happy 4th to all.
Does anyone have a procedure on what to do when a tissue processor fails or 
alarms.  I want to learn more about the science behind tissue processing so I 
know what to do when the machine fails. This happened to a friend recently and 
I want to prevent my tissues/biopsies from being ruined.
Thanks.
Garrey

Sent from my iPhone
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[Histonet] Lab assistant/histotech

[Samantha Golden, HT(ASCP) via Histonet]

I was curious if there is a source, publication, report, study, general 
information, regarding the use of lab assistants in the histology lab.

I would like the know the percentage of labs that utilize assistants versus 
labs with techs only. I would also like to know that for labs that do utilize 
assistants, what duties do they perform that the techs are exempt from.

I’m simply curious to see how other labs have workflow setup.

Thanks for any insight.

Samantha


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