[Histonet] Frozen tissue from Immunofluorescence

[O'Neil, Beth A. via Histonet]

Would anyone be willing to share with me what you do with residual frozen 
tissue from immunofluorescence testing or frozen tissue remaining from muscle 
biopsy?  CAP requirements only address what you do with residual frozen tissue 
remaining from intraoperative consultations.  We keep the frozen tissue from IF 
in our -70 C freezer and then periodically dispose of the oldest cases in order 
to make room for the newer.  Does anyone out there actually take the frozen 
tissue, after a certain period of time, thaw it out and then process into a 
FFPE block for storage?

Beth ONeil
beth.one...@wvumedicine.org

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[Histonet] Frozen tissue

[Anne Murvosh via Histonet]

I'm testing a faster Mart 1 for melanoma on Mohs specimens. Its been a while 
since I've done any frozen tissue IHC stains and I can't remember the fixation 
to keep it viable for testing the next day, or same day but later on. Will it 
hold up if fixed in acetone and dried or do I need to refrigerate or freeze? 
Also how long do they last,  I can't remember the protocol. Thanks Anne HT
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[Histonet] frozen tissue training

[Amy Lee]

 Hello histonet,
I am looking for a frozen tissue training session for my new employee. It 
includes frozen tissue harvest, storing, sectioning, etc. Could any one 
recommend a training session inside US?
Thanks,
Amy
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RE: [Histonet] frozen tissue training

[Jacox, Robert A.]

Amy,

I am not sure what type of tissue you are cutting but the MOHs society has a 
nice training course. 

Fundamentals of Mohs Surgery for Physicians and Mohs Technicians 

Thursday, November 5 – Sunday, November 8, 2015

Robert Jacox
Manager, Global Tactical Marketing
Anatomic Pathology

Thermo Fisher Scientific
Tel: 269-544-5651 l Mobile: 269-598-0747
robert.ja...@thermofisher.com l www.thermoscientific.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amy Lee
Sent: Monday, March 02, 2015 2:32 PM
To: histonet
Subject: [Histonet] frozen tissue training

 Hello histonet,
I am looking for a frozen tissue training session for my new employee. It 
includes frozen tissue harvest, storing, sectioning, etc. Could any one 
recommend a training session inside US?
Thanks,
Amy
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[Histonet] Frozen tissue/OCT Vials

[Cooper, Brian]

It's almost Friday Histonetters!

What kinds of containers are you storing your OCT embedded samples in?  We're 
currently using snap top vials for all of our frozen samples that have a 
tendency to pop their lids when removed from our LN2 freezers!   Wondering if 
there are some type of screw top vials that will suit our purposes.  I know we 
will likely have to change the boxes we store our vials in as well, but that's 
par for the course.  Any help will be greatly appreciated!

Thanks,

Brian D. Cooper, HT (ASCP)CM | Histology Supervisor
Department of Pathology and Laboratory Medicine
Children's Hospital Los Angeles
4650 Sunset Blvd MS#43- Los Angeles, CA 90027
Ph: 323.361.3357 Pager: 213-209-0184
bcoo...@chla.usc.edumailto:bcoo...@chla.usc.edu



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message.  

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[Histonet] Frozen Tissue Retention

[Bradley, Joshua, D]

I am curious as to the frozen tissue retention policies in other institutions, 
especially in children's facilities.  How long are samples considered 
diagnostically viable and relevant to patient care?  What is done with the 
tissue when the time limit is reached?

Our facility is interested in using old samples in research applications 
(unidentified of course).  We currently have a retention policy of 10 years, 
but wanted to gauge how other facilities approach this matter.

Thanks for any help that can be provided.

Joshua D. Bradley, B.S., HT(ASCP)CM
Clinical Lab Supervisor-Histology
Children's Mercy Hospital
Kansas City, MO



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[Histonet] Frozen tissue detachment

[Marie Madsen]

Hi everyone,

We're having trouble with frozen tissue detachment (mouse aortic root).
Our procedure is as follows:

1. Fresh heart into NBF 4% (Lilly's) 24h, fridge (+4)
2. OCT (tissue-tek) 2h, fridge (+4)
3. Quickly frozen in icecold isopentan
4. Freezer (-20) until sectioned at 10 µm in cryostat (at approx -20)
5. RT one to several hours before being put back into the freezer.

We use Superfrost glass slides.

The *only* thing I can think of is wrong, is that the sections have been 
thawed/frozen several times before being used for staining (we do that to find 
the best section for our stainings) -do you think that's the problem?

Another thing could be that after being sectioned we don't have a specific 
amount of time and temperature at which the sections should dry..

I found out recently regarding my IHC protocol, that removing the OCT with 70% 
EtOH (10 min = dry 20 min) makes the tissue stick better than removing it with 
dH2O or TBS..but then the specific staining also became much more faded..any 
ideas what else to do?

Sorry about all the questions -I'm relatively new to this field, but find it 
very interesting.

Best, Marie
  
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[Histonet] Frozen Tissue Protocol

[Sheila Fonner]

Hi everyone,

Yesterday I asked for help with modifying my protocols to stain HSVI, HSVII,
and Zoster.  I appreciate everyone's help.  Now I am back for more info.  I
deleted the depar. and cell conditioning steps and I got specific staining
with little background.  There was just one problem.the pathologist says
that the leukocytes are reacting with the alk. phos on all of the slides.
Does anyone know if a blocking step would help in this case?  Just a
reminder - I am using the Ventana Ultra platform with Ultraview Red
Detection.  Thanks again Histoland.  You never let me down! J

Sheila

 

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RE: [Histonet] Frozen Tissue Protocol

[Sheila Fonner]

Paula,

 

Where would I insert this step in my protocol if I am staining on the
Ventana Ultra?  Can I do it by hand after the slides are finished?  Thanks
for your help.

 

Sheila

 

 

From: Paula Pierce [mailto:cont...@excaliburpathology.com] 
Sent: Wednesday, August 17, 2011 11:14 AM
To: Sheila Fonner
Subject: Re: [Histonet] Frozen Tissue Protocol

 

You are seeing endogenous phosphatase, not endogenous peroxidase. Use 10%
acetic acid for 1-2 minutes.
 

Paula K. Pierce, HTL(ASCP)HT

President

Excalibur Pathology, Inc.

631 N Broadway

Moore, OK 73160

405-759-3953 Lab

405-759-7513 Fax

www.excaliburpathology.com http://www.excaliburpathology.com/ 

 

 

  _  

From: Sheila Fonner sfon...@labpath.com
To: histonet@lists.utsouthwestern.edu
Sent: Wed, August 17, 2011 9:45:18 AM
Subject: [Histonet] Frozen Tissue Protocol

Hi everyone,

Yesterday I asked for help with modifying my protocols to stain HSVI, HSVII,
and Zoster.  I appreciate everyone's help.  Now I am back for more info.  I
deleted the depar. and cell conditioning steps and I got specific staining
with little background.  There was just one problem.the pathologist says
that the leukocytes are reacting with the alk. phos on all of the slides.
Does anyone know if a blocking step would help in this case?  Just a
reminder - I am using the Ventana Ultra platform with Ultraview Red
Detection.  Thanks again Histoland.  You never let me down! J

Sheila



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Re: [Histonet] Frozen tissue question

[Emily Sours]

From the research point of view, I've heard of people not fixing tissue
before they section it. Since I work with embryonic tissue (which is mostly
water!), we always fix our tissue. It definitely is better to not fix tissue
when you want to stain with antibodies because anything you do to the sample
might affect the antigens.  However, since it's impossible to section
unfixed tissue, you have to fix it somehow.
Our lab once tried to section snap frozen unfixed chick embryo because
another lab (our Sauron, if you will) had said that's the best way to get
antibody staining.  We, however, could not get the tissue to section
properly.  How that other lab did it is beyond me.
Either way, there's never a clear yes or no with protocols, it'll always
change due to what you're sectioning.

Emily

A great book should leave you with many experiences, and slightly exhausted.
You should live several lives while reading it.
-William Styron



On Thu, Jun 23, 2011 at 1:47 PM, Nicole Tatum nic...@dlcjax.com wrote:

 I run a Mohs lab that processes skin by frozen section. There is no need
 to use any fixative before hand. But, if you are looking for melanoma or
 melanocytes, freezing can cause artifact and make it difficult to read
 slides. Limit the amount of nitrogen you use to freeze the specimen. Some
 places use alcohol as a fixative after the slide is cut. It will for all
 purposes remove the water continent from the tissue which will preserve
 tissue. Hope this helps.

 Nicole Tatum HT ASCP





  We have a new doctor in our lab who swears that all frozen tissue must
  be fixed in formalin with a subsequent sucrose treatment before freezing
  in OCT because not fixing it will cause the structures to be distorted
  and you can't get good antibody attachment. In my previous experience,
  we have done this with tissue that came from an animal that was perfused
  with formalin before the tissue was removed. However, the majority of my
  previous frozen specimens were flash frozen in OCT and fixed after
  sectioning. It is also my understanding that fixation in formalin can
  cause poor antibody detection because of cross-linking caused by the
  formalin. I would like to hear some other opinions on this please. The
  particular specimen we will be working with is skin.
 
  Thanks in advance,
  Joel Reichensperger
 
  --
  Joel Reichensperger
  Researcher II
  Southern Illinois University
  Plastic Surgery Institute
  jreichensper...@siumed.edu
  217-545-7309 (Office)
  217-545-1824 (Fax)
 
 
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[Histonet] Frozen tissue question

[Joel Reichensperger]

We have a new doctor in our lab who swears that all frozen tissue must 
be fixed in formalin with a subsequent sucrose treatment before freezing 
in OCT because not fixing it will cause the structures to be distorted 
and you can't get good antibody attachment. In my previous experience, 
we have done this with tissue that came from an animal that was perfused 
with formalin before the tissue was removed. However, the majority of my 
previous frozen specimens were flash frozen in OCT and fixed after 
sectioning. It is also my understanding that fixation in formalin can 
cause poor antibody detection because of cross-linking caused by the 
formalin. I would like to hear some other opinions on this please. The 
particular specimen we will be working with is skin.


Thanks in advance,
Joel Reichensperger

--
Joel Reichensperger
Researcher II
Southern Illinois University
Plastic Surgery Institute
jreichensper...@siumed.edu
217-545-7309 (Office)
217-545-1824 (Fax)


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Re: [Histonet] Frozen tissue question

[Rene J Buesa]

Your new doctor is absolutely wrong. You do not need to formalin fix or 
sucrose wash before freezing the specimen in OCT. You can go directly from the 
fresh specimen → OCT freezing → cutting.
You are partially right about the cross-linking of the antigens; only 
partially because cross-linking requires about 20 hours and you will never 
wait so long to make the FS.
In any event, it is not necessary to do what your new doctor says; it is 
evident that s/he is really new.
René J.
 
 

--- On Thu, 6/23/11, Joel Reichensperger jreichensper...@siumed.edu wrote:


From: Joel Reichensperger jreichensper...@siumed.edu
Subject: [Histonet] Frozen tissue question
To: histonet@lists.utsouthwestern.edu
Date: Thursday, June 23, 2011, 10:12 AM


We have a new doctor in our lab who swears that all frozen tissue must be fixed 
in formalin with a subsequent sucrose treatment before freezing in OCT because 
not fixing it will cause the structures to be distorted and you can't get good 
antibody attachment. In my previous experience, we have done this with tissue 
that came from an animal that was perfused with formalin before the tissue was 
removed. However, the majority of my previous frozen specimens were flash 
frozen in OCT and fixed after sectioning. It is also my understanding that 
fixation in formalin can cause poor antibody detection because of cross-linking 
caused by the formalin. I would like to hear some other opinions on this 
please. The particular specimen we will be working with is skin.

Thanks in advance,
Joel Reichensperger

-- Joel Reichensperger
Researcher II
Southern Illinois University
Plastic Surgery Institute
jreichensper...@siumed.edu
217-545-7309 (Office)
217-545-1824 (Fax)


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Re: [Histonet] Frozen tissue question

[Nicole Tatum]

I run a Mohs lab that processes skin by frozen section. There is no need
to use any fixative before hand. But, if you are looking for melanoma or
melanocytes, freezing can cause artifact and make it difficult to read
slides. Limit the amount of nitrogen you use to freeze the specimen. Some
places use alcohol as a fixative after the slide is cut. It will for all
purposes remove the water continent from the tissue which will preserve
tissue. Hope this helps.

Nicole Tatum HT ASCP





 We have a new doctor in our lab who swears that all frozen tissue must
 be fixed in formalin with a subsequent sucrose treatment before freezing
 in OCT because not fixing it will cause the structures to be distorted
 and you can't get good antibody attachment. In my previous experience,
 we have done this with tissue that came from an animal that was perfused
 with formalin before the tissue was removed. However, the majority of my
 previous frozen specimens were flash frozen in OCT and fixed after
 sectioning. It is also my understanding that fixation in formalin can
 cause poor antibody detection because of cross-linking caused by the
 formalin. I would like to hear some other opinions on this please. The
 particular specimen we will be working with is skin.

 Thanks in advance,
 Joel Reichensperger

 --
 Joel Reichensperger
 Researcher II
 Southern Illinois University
 Plastic Surgery Institute
 jreichensper...@siumed.edu
 217-545-7309 (Office)
 217-545-1824 (Fax)


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