[Histonet] grad student problem

[Greg Dobbin via Histonet]

I can't speak to the the T-cell receptor sites but the only option for
getting decent sections at this point is option number two. In the future,
(if there applicable) the cut surface of a cryosectioned block can be
recovered with OCT, frozen, and stored in an air-tight whirl-pak or ziplock
bag in the freezer for a little longer to avoid freezer burn.
Cheers,
Greg

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Re: [Histonet] Grad Student Problem

[Elizabeth Chlipala via Histonet]

You can also thaw and refreeze we have done that before.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
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(303) 682-3949 office
(303) 682-9060 fax
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-Original Message-
From: Terri Braud via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, January 04, 2017 12:16 PM
To: 'histonet@lists.utsouthwestern.edu'
Cc: 'r...@psu.edu'
Subject: Re: [Histonet] Grad Student Problem

My response:
My first question is what temperature are the blocks that are too brittle? And 
at what temperature are you trying to cut?  Blocks stored in cryofreezers at 
-70o C or less are far too cold to cut without brittleness.  My suggestion 
would be to pull the blocks and put them into the cryostat at -18o C for at 
least 6 hours to acclimate to that temp, then try to cut and stain for IF.

If that doesn't work, then I would thaw in formalin and process as routine 
tissue for formalin fixed paraffin embedded.  The process that your student 
described in the 2nd step is not a complete, or valid process.
Depending on the T-Cell markers they are trying to demonstrate, one can 
successfully use standard IHC with appropriate clones, or with usually less 
success, use a modified IF stain procedure for FFPE sections. Since there are a 
"ba-zillion" T-cell markers, any further details are very dependent on the 
markers and the condition of the tissue.
Sincerely, Terri Braud

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal
Today's Topics:

   1. grad student problem (Roberta Horner)
Message: 1
Date: Tue, 3 Jan 2017 18:05:15 +
From: Roberta Horner <r...@psu.edu>
I got the following from a grad student here at Penn State. I am not sure how 
to solve his problem if possible. Does anyone have any suggestions I can 
forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University

"I am having some difficulties sectioning mouse tumor samples for 
immunofluorescent analysis.  We originally went the OCT route because we are 
staining for T cell markers and were worried that the heating that occurs 
during paraffin embedding would compromise the T cell receptor.  The samples 
are a little old, but we are hoping to section and stain for immune cell 
infiltrates.  When sectioning with the cryostat, the tissue and OCT is quite 
brittle and the sample is not intact enough to transfer to a slide. Two 
colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh 
OCT media.  I've tried this technique on a few practice samples and the new OCT 
media seems to be less brittle and I'm able to get tissue on a slide, however 
the tissue itself still seems to be poor with either freeze or sectioning 
artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin 
overnight, place in 70% EtOH, then paraffin embed.  Section on a microtome.  
Check the fluorescently labelled antibody data sheet to see if paraffin 
embedding interferes with binding.  Try to stain and see what happens.

I was hesitant to try the second suggestion because I have found no protocol 
that takes tissue originally stored in OCT blocks and subsequently redirects 
them to formalin and paraffin for microtome sectioning.  If you have any 
recommendations on how to move forward and section these difficult samples, or 
know anyone at the diagnostics lab or at Penn State that could help, that would 
be much appreciated!"

*


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Re: [Histonet] Grad Student Problem

[Terri Braud via Histonet]

My response:
My first question is what temperature are the blocks that are too brittle? And 
at what temperature are you trying to cut?  Blocks stored in cryofreezers at 
-70o C or less are far too cold to cut without brittleness.  My suggestion 
would be to pull the blocks and put them into the cryostat at -18o C for at 
least 6 hours to acclimate to that temp, then try to cut and stain for IF.

If that doesn't work, then I would thaw in formalin and process as routine 
tissue for formalin fixed paraffin embedded.  The process that your student 
described in the 2nd step is not a complete, or valid process.
Depending on the T-Cell markers they are trying to demonstrate, one can 
successfully use standard IHC with appropriate clones, or with usually less 
success, use a modified IF stain procedure for FFPE sections. Since there are a 
"ba-zillion" T-cell markers, any further details are very dependent on the 
markers and the condition of the tissue.
Sincerely, Terri Braud

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal
Today's Topics:

   1. grad student problem (Roberta Horner)
Message: 1
Date: Tue, 3 Jan 2017 18:05:15 +
From: Roberta Horner 
I got the following from a grad student here at Penn State. I am not sure how 
to solve his problem if possible. Does anyone have any suggestions I can 
forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University

"I am having some difficulties sectioning mouse tumor samples for 
immunofluorescent analysis.  We originally went the OCT route because we are 
staining for T cell markers and were worried that the heating that occurs 
during paraffin embedding would compromise the T cell receptor.  The samples 
are a little old, but we are hoping to section and stain for immune cell 
infiltrates.  When sectioning with the cryostat, the tissue and OCT is quite 
brittle and the sample is not intact enough to transfer to a slide. Two 
colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh 
OCT media.  I've tried this technique on a few practice samples and the new OCT 
media seems to be less brittle and I'm able to get tissue on a slide, however 
the tissue itself still seems to be poor with either freeze or sectioning 
artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin 
overnight, place in 70% EtOH, then paraffin embed.  Section on a microtome.  
Check the fluorescently labelled antibody data sheet to see if paraffin 
embedding interferes with binding.  Try to stain and see what happens.

I was hesitant to try the second suggestion because I have found no protocol 
that takes tissue originally stored in OCT blocks and subsequently redirects 
them to formalin and paraffin for microtome sectioning.  If you have any 
recommendations on how to move forward and section these difficult samples, or 
know anyone at the diagnostics lab or at Penn State that could help, that would 
be much appreciated!"

*


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Re: [Histonet] grad student problem

[Loralei Dewe via Histonet]

Completely agree...it sounds like the tissue got freeze dried and I doubt
if any staining you might get after lots of work will be completely
reliable.

Loralei



On Jan 3, 2017 11:02 AM, "Caroline Miller via Histonet" <
histonet@lists.utsouthwestern.edu> wrote:

> I hate to say it but I think these tissues are toast. It seems they had bad
> fixation or handling to start with, and I would question whether the
> tissues were left out to dry before fixation or freezing. Or whether they
> got fixed and then sucrose infiltrated (which is my method of choice if the
> antibody allows)
>
> Also - what type of tissues? If it is lymph nodes then maybe there is too
> much fatty tissue and that is why it is not sectioning.
>
> I have had a hard time with T-cell markers in paraffin so I do see the
> hesitation in paraffin embedding. If you do go that route then certainly
> thaw in formalin, not straight water.
>
> Not much help, sorry! But sometimes it is just better to go back and plan
> the experiment properly in the first place than trying to mess around with
> old bits of tissue - because even if you could get it on the slide, would
> you really trust the results of the immunostaining???
>
> Happy New Year All!
>
> yours,
> mills
>
> On Tue, Jan 3, 2017 at 10:05 AM, Roberta Horner via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > I got the following from a grad student here at Penn State. I am not sure
> > how to solve his problem if possible. Does anyone have any suggestions I
> > can forward?
> > Roberta Horner
> > Animal Diagnostic Lab
> > Penn State University
> >
> > "I am having some difficulties sectioning mouse tumor samples for
> > immunofluorescent analysis.  We originally went the OCT route because we
> > are staining for T cell markers and were worried that the heating that
> > occurs during paraffin embedding would compromise the T cell receptor.
> The
> > samples are a little old, but we are hoping to section and stain for
> immune
> > cell infiltrates.  When sectioning with the cryostat, the tissue and OCT
> is
> > quite brittle and the sample is not intact enough to transfer to a slide.
> > Two colleagues have given the following suggestions:
> > 1. Thaw the OCT blocks, remove the tissue, and place in a new block with
> > fresh OCT media.  I've tried this technique on a few practice samples and
> > the new OCT media seems to be less brittle and I'm able to get tissue on
> a
> > slide, however the tissue itself still seems to be poor with either
> freeze
> > or sectioning artifacts.
> > 2. Thaw the OCT blocks in water, remove the tissue and place in formalin
> > overnight, place in 70% EtOH, then paraffin embed.  Section on a
> > microtome.  Check the fluorescently labelled antibody data sheet to see
> if
> > paraffin embedding interferes with binding.  Try to stain and see what
> > happens.
> >
> > I was hesitant to try the second suggestion because I have found no
> > protocol that takes tissue originally stored in OCT blocks and
> subsequently
> > redirects them to formalin and paraffin for microtome sectioning.  If you
> > have any recommendations on how to move forward and section these
> difficult
> > samples, or know anyone at the diagnostics lab or at Penn State that
> could
> > help, that would be much appreciated!"
> >
> > ___
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
>
>
>
> --
> Caroline Miller (mills)
> Director of Histology
> 3Scan.com
> 415 2187297
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>
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Re: [Histonet] grad student problem

[Caroline Miller via Histonet]

I hate to say it but I think these tissues are toast. It seems they had bad
fixation or handling to start with, and I would question whether the
tissues were left out to dry before fixation or freezing. Or whether they
got fixed and then sucrose infiltrated (which is my method of choice if the
antibody allows)

Also - what type of tissues? If it is lymph nodes then maybe there is too
much fatty tissue and that is why it is not sectioning.

I have had a hard time with T-cell markers in paraffin so I do see the
hesitation in paraffin embedding. If you do go that route then certainly
thaw in formalin, not straight water.

Not much help, sorry! But sometimes it is just better to go back and plan
the experiment properly in the first place than trying to mess around with
old bits of tissue - because even if you could get it on the slide, would
you really trust the results of the immunostaining???

Happy New Year All!

yours,
mills

On Tue, Jan 3, 2017 at 10:05 AM, Roberta Horner via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I got the following from a grad student here at Penn State. I am not sure
> how to solve his problem if possible. Does anyone have any suggestions I
> can forward?
> Roberta Horner
> Animal Diagnostic Lab
> Penn State University
>
> "I am having some difficulties sectioning mouse tumor samples for
> immunofluorescent analysis.  We originally went the OCT route because we
> are staining for T cell markers and were worried that the heating that
> occurs during paraffin embedding would compromise the T cell receptor.  The
> samples are a little old, but we are hoping to section and stain for immune
> cell infiltrates.  When sectioning with the cryostat, the tissue and OCT is
> quite brittle and the sample is not intact enough to transfer to a slide.
> Two colleagues have given the following suggestions:
> 1. Thaw the OCT blocks, remove the tissue, and place in a new block with
> fresh OCT media.  I've tried this technique on a few practice samples and
> the new OCT media seems to be less brittle and I'm able to get tissue on a
> slide, however the tissue itself still seems to be poor with either freeze
> or sectioning artifacts.
> 2. Thaw the OCT blocks in water, remove the tissue and place in formalin
> overnight, place in 70% EtOH, then paraffin embed.  Section on a
> microtome.  Check the fluorescently labelled antibody data sheet to see if
> paraffin embedding interferes with binding.  Try to stain and see what
> happens.
>
> I was hesitant to try the second suggestion because I have found no
> protocol that takes tissue originally stored in OCT blocks and subsequently
> redirects them to formalin and paraffin for microtome sectioning.  If you
> have any recommendations on how to move forward and section these difficult
> samples, or know anyone at the diagnostics lab or at Penn State that could
> help, that would be much appreciated!"
>
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



-- 
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297
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[Histonet] grad student problem

[Roberta Horner via Histonet]

I got the following from a grad student here at Penn State. I am not sure how 
to solve his problem if possible. Does anyone have any suggestions I can 
forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University

"I am having some difficulties sectioning mouse tumor samples for 
immunofluorescent analysis.  We originally went the OCT route because we are 
staining for T cell markers and were worried that the heating that occurs 
during paraffin embedding would compromise the T cell receptor.  The samples 
are a little old, but we are hoping to section and stain for immune cell 
infiltrates.  When sectioning with the cryostat, the tissue and OCT is quite 
brittle and the sample is not intact enough to transfer to a slide. Two 
colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh 
OCT media.  I've tried this technique on a few practice samples and the new OCT 
media seems to be less brittle and I'm able to get tissue on a slide, however 
the tissue itself still seems to be poor with either freeze or sectioning 
artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin 
overnight, place in 70% EtOH, then paraffin embed.  Section on a microtome.  
Check the fluorescently labelled antibody data sheet to see if paraffin 
embedding interferes with binding.  Try to stain and see what happens.

I was hesitant to try the second suggestion because I have found no protocol 
that takes tissue originally stored in OCT blocks and subsequently redirects 
them to formalin and paraffin for microtome sectioning.  If you have any 
recommendations on how to move forward and section these difficult samples, or 
know anyone at the diagnostics lab or at Penn State that could help, that would 
be much appreciated!"

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