[Histonet] Attention Daniel Blackburn about plastics embedding

[Gayle Callis via Histonet]

You wrote: 

 

My lab hopes to get into plastic sectioning.  We need to be able to process
tissue pieces as large and thick as possible, but see that the largest
embedding molds for JB4 are only 13x19mm by 5mm deep.   We have two
questions:  (1) Do any of the available media (plastic or resins) allow one
to embed and section large  pieces (for example eggs with dimensions of 2 cm
or larger)?   (2) Is a special microtome (such as a retracting microtome)
needed?   Our reason for considering plastic is that we must section yolk,
which splits out of standard paraffin during sectioning. Any advice is
appreciated. -- Daniel Blackburn, Trinity College 

 



Glycol methacrylate is not designed for samples as large at 2 cm.  However,
I have one publication from an old Stain Technology for large
chondro-osseous (sp?) sample.   I also have a protocol from a lady who used
water based processing with GMA since it is water miscible.   This was a
protocol lipids which are removed by organic solvents.   You might be able
to develop a protocol with your 2 cm samples.   Kits are expensive and I
have information on making up GMA in house.   Hope you have a good fume
hood!!  As you can see from another post today, GMA contains sensitizing
chemicals, and a carcinogen.  It will require double gloving with nitrile
gloves, and other personal safety gear.   The fume hood is an absolute must
have. 

 

You will need a powerful microtome, i.e. Leica 2250 or 2265 or equivalent,
with very sharp knives, maybe even tungsten carbide.  We used glass knives
on a JB-4 microtome but I have a colleague who used a 2250 and disposable
microtome blades, but you blocks are pretty large.   You may want to
consider using Peel away molds which come in several sizes.   With your size
sample, infiltration and polymerization will be tricky.  You have to seal
air away from the top of molds in order to get the blocks to polymerize.  I
think people have used plastic wrap over the top of molds to exclude air
although our metal blocks fit in the embedding molds snugly and we did an
old school method to exclude air.  Melted paraffin around outside of molds,
a messy but effective air block.   

 

Please contact me personally and I will send this this information to you.
I think you can do this with a reply to all after reading this message.   

 

Take care

Gayle Callis HTL, HT, MT(ASCP)

GCallis Histology Service LLC

Bozeman MT/USA  

 

 

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Re: [Histonet] glycol methylacrylate GMA enzyme and IHC

[Gayle Callis via Histonet]

You wrote: 

Hi all,

 

I am going to be exploring some tissue embedding using Glycolmethacrylate

(GMA), as I read that it can preserve enzyme function in some cases and

potentially can be used for IHC. However, information is a bit scant and

can be contradictory at times, to say the least.

 

If anyone has any experience doing any histological or IHC staining on GMA

embedded tissue, or knows of anywhere I could get protocols from or any

papers that are a bit more recent (I'm mostly finding 90s, early 00s),

pleas let me know, I would really appreciate any resources I can find!

 

-- 

Casey Berridge

 


**

GMA references from the 80’s and 90’s pretty much remain current for GMA.
Be sure to do a literature search in J Histochem Cytochem (JHC.org) to see
if there are more recent publications.  Google Scholar was not turning up
much for recent years.

 

We worked with GMA for many years but never for IHC.   There are many
considerations when using GMA both good and bad with emphasis on the
negative side of things from my point of view.   We had success when
studying some single cell protozoa, including Cryptosporidia in a research
setting.   

 

There is one publication in the old Stain Technology, now Biotechnic &
Histochemistry using GMA for successful enzyme staining.  Namba M et al.
Improvement in histochemical demonstration of esterase in glycol
methacrylate tissue sections by cold temperature embedding in glycol
methacrylate. 1983 58(4):207.

 

Several things about GMA. 

 

Requires a fume hood in order to work with toxic and carcinogenic chemicals.
Glycol methacrylate is sensitizing and several colleagues are so allergic to
fumes after working with this plastic over several years, they can’t be in
the same room where GMA is being worked with.  Double gloving is advisable,
and wearing safety glasses is a must since the sections are small and can
fly into an eye (know of this happening) which is not a good situation.
There should be no skin contact with the plastic, nor breathing the n, n, di
methylaniline, a carcinogen.Controlling polymerization can be a problem
unless you place embedding molds on top if ice, and cooling the embedding
mixture with ice water.   The polymerization is exothermic, and actually as
blocks polymerize gets uncomfortably hot which may be damaging to enzymes
and sensitive antigens although the heat can be dispersed.   

 

Samples cannot be any thicker than 2 mm, with 1 mm X 1 mm is recommended.
This plastic was first used for liver needle biopsies.Polymerization for
larger samples is hard to control as is the infiltration by this plastic
hence smaller, thinner samples.   Sectioning is commonly done with glass
knives although tungsten carbide knives work, and I know of one group using
disposable blades with a Leica 2255 model microtome.More powerful
microtomes i.e. Leica 2650 or equivalent works best.  We had a JB-4
microtome with a special block holder to accommodate the metal
“chucks”/block holders sold by Polysciences.   The metal block holders were
a better heat sink to disperse heat of polymerization. Sections are
generally no thicker than 1 to 3 µm, and were wonderful when studying single
cell protozoa.  We never used GMA for more routine tissue sections although
it was popular for bone biopsies in clinical labs over the many years.   I
personally found it labor intensive, and expensive for our projects although
the staining results for H, PAS-H and some other special stains very nice.


 

Routine stains can be used, including PAS-H, H, Massons trichrome with a
modified method, and others.   IHC will not work well, even with JB-4
Immunobed.  GMA, once polymerized, cannot be removed from the section.
Immunobed is probably just a looser matrix than JB-4 and some people have
success.   GMA  plastic is less hydrophobic but still will not allow  large
immunoglobulins to reach antigenic sites.   There has been some success with
IHC but in general, GMA is not the ideal embedding media for immunostaining.
Neil Hand worked with Poly methylmethacrylate for IHC since the plastic can
be completely removed from a thin section, followed by stringent HIER using
a pressure cooker.   PMMA is another world for processing,  sectioning and
staining.  

 

When doing H, the staining protocol is different from paraffin section
staining.   If you do an extensive, time intensive search on Histonet, there
are many discussions about GMA staining both for routine and IHC.  

 

You can buy kits, JB-4 and Technovits.   The JB-4 discolors over the years
to a dark tea/brown color making it more difficult to see the tissue while
Technovits remains clear.   When you cut sections, you work with one section
at a time, not a ribbon.   

 

I have a huge file on GMA collected from the early 70’s all the way to
current years.  If you reply to me personally, I 

[Histonet] Low temperature freezer

[Tamara Howard via Histonet]

Richard -


We learned last year (the hard way) that chest freezers are more likely to 
survive a flood than are uprights, if that is any help! The compressors tend to 
be near the top of the unit in the chest freezers. I think the footprint may be 
the main deciding factor for many labs; we just bought an upright to replace 
one that the flood destroyed, only because we don't have space to put in a 
chest freezer with the same storage capacity.


Tamara


...
Tamara Howard
Dept. of Cell Biology & Physiology
University of New Mexico
Albuquerque, NM

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Re: [Histonet] Prefilled formalin containers

[Kienitz, Kari via Histonet]

I have had this annoying experience a few times over the last 20+ years.  It 
seems to me the breakdown is with quality control at the manufacturer.  Most 
recently, 1 out of 5 containers  came back to the lab leaking.  I put pressure 
on my vendor, who put pressure on his vendor.  Unfortunatly, his vendor really 
didn't take the situation seriously.  Even after explaining the dangers to lab 
personnel as well as patients being exposed to formalin they did nothing.

I feel fortunate the histology supply vendor I deal with values our business. 
He went and found a new vendor/manufacturer that has supplied us with 
containers that don't leak and so far they understand the health hazzard and 
the annoyance of what to many may seem trivial.



Kari Kienitz HT, (ASCP)
Histology Laboratory
Gastroenterology-EAST
The Oregon Clinic
 NE 99th Ave
Portland, OR  97220
503.935.8311
kkien...@orclinic.com




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From: Vickroy, James via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Monday, July 18, 2016 9:33 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Prefilled formalin containers

This is one of the things that comes up every couple of years.   Leaky 
prefilled formalin containers.   I know we have all dealt with nurses or 
physicians that can't put the lid on correctly or put the label on the threads 
of the containers.  Some of the new designs have a "clicking lid" when they are 
supposed to be sealed.   My experience with the "clicking lids" are that some 
vendors have lids that the "clicker" breaks off as soon as you unscrew it so 
obviously it doesn't help when putting the lid back on.   Another vendor that 
has the "clicking lid" does not have the "clicker" break off when you unscrew 
and screw but the containers still leak.

It seems to me that someone could come up with accost-effective prefilled 
formalin container that does not leak (of course provided the lids was put on 
straight).   I would be interested in other's experiences with this elemental 
yet extremely annoying issue. It seems like if we can make new automated 
electronic instruments someone might be able to make affordable container that 
doesn't leak.

Jim

Jim Vickroy
Histology Manager
Springfield Clinic, Main Campus, East Building
1025 South 6th Street
Springfield, Illinois  62703
Office:  217-528-7541, Ext. 15121
Email:  jvick...@springfieldclinic.com



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Re: [Histonet] expiration dates

[Elizabeth Chlipala via Histonet]

Linda

We have in our reagent policy that any chemical that does not come with an 
expiration date - we assign it a 5 year from receipt expiration date.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Blazek, Linda via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, July 19, 2016 10:35 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] expiration dates

Does anyone see an expiration date printed on their can of Freeze Spray?
I was told by the company I purchase mine from that it was not needed because 
it was "not a medical device".

Thanks
Linda

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Re: [Histonet] expiration dates

[Bell, Lynne via Histonet]

My can of Fisherbrand Freeze'it does not have an expiration date on it but I 
have Cytocool II from Richard-Allan Scientific does have one (it's printed on 
the bottom of the can).


Lynne Bell, HT (ASCP)
Histology Team Leader
Central Vermont Medical Center
130 Fisher Road
Berlin, VT  05641
(802)371-4923







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[Histonet] expiration dates

[Blazek, Linda via Histonet]

Does anyone see an expiration date printed on their can of Freeze Spray?
I was told by the company I purchase mine from that it was not needed because 
it was "not a medical device".

Thanks
Linda

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[Histonet] Blog Post (with Explanation)

[Lester Raff MD via Histonet]

The linked blog is not about lab/histology, but a histonetter Downsize, Maybe 
subscriber who read it suggested that it could perhaps  be useful for h. pylori 
and worth a post-so here goes.

http://www.chicagonow.com/downsize-maybe/2016/07/we-take-a-shine-to-yinmn-its-the-new-blue/

Lester J. Raff, MD MBA
UroPartners
Medical Director Of Laboratory
2225 Enterprise Dr. Suite 2511
Westchester, Il 60154
Tel: 708-486-0076
Fax: 708-492-0203

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[Histonet] Plastic embedding and Microtomy

[Cassie P. Davis via Histonet]

Hi Daniel,

I haven't seen plastics embedding and microtomy since the mid 90's. 
When I was at CCHS in Newark, DE we used to do our bone marrow biopsies in 
them. At that time there were molds big enough to embed a penny in.

Yes, you need a different microtome to section these specimens. We had a 
glass knife micotome. We had to score and break glass knives at least once a 
week. This is a little dangerous and quite tempermental. The edge on the glass 
knife didn't seem to last more than 5 days and not everybody was good at making 
the glass knives. We adventually, reverted back to paraffin a few years before 
I left ('99). If I remember correctly, IHC does not perform well on the 
"plastics" specimens which is why we would typically process and embed in 
paraffin. We would routinely perform trichrome,PAS and retic on the plastic 
sections as well as H I wish I could offer you more information than this.


Cassandra Davis
Histology Technician
Anatomical Pathology Laboratory
Saint Francis Healthcare
701 N. Clayton Street
Wilmington,DE 19805
Office:  302-575-8095
Email:  cda...@che-east.org
www.saintfrancishealthcare.org

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