Re: [Histonet] Paraformaldehyde Fixed Tissue

[Ana Maluenda via Histonet]

Hi Sandra,

I haven't myself particularly worked with brain and spinal cord, but majority 
of my protocols in my old job used fixation in 4% PFA (24 hours at 4-8oC) and 
routinely process (or transfer to graded EtOH, if not processing immediately).
However, our routine process didn't include a first step in 10% NBF, since PFA 
plays the role of fixation. Therefore, after PFA, we would have 70% EtOH, 80% 
EtOH, 95% ETOH, 2x EtOH the Xylenes and wax [assuming you are referring to 
FFPE?].
Also mouse tissue can be small and delicate, so I remember running liver, 
spleen, kidney and thymus (soft tissues I worked with) in a short cycle 
(similar to what I would do for biopsies).

Hope this helps!

Kind regards,

Ana


Ana Maluenda
Research Assistant
Atherothrombosis and Vascular Biology Laboratory

Baker Heart and Diabetes Institute
75 Commercial Road, Melbourne VIC 3004
P (03) 8532 1359 E ana.malue...@baker.edu.au W www.baker.edu.au

-Original Message-
From: Sandra Cheasty [mailto:sandra.chea...@wisc.edu]
Sent: Tuesday, 29 August 2017 1:30 AM
To: Histonet (histonet@lists.utsouthwestern.edu) 

Subject: [Histonet] Paraformaldehyde Fixed Tissue

Hi all,
We are having difficulty sectioning mouse tissue, (brain, 
spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone 
had issues with paraformaldehyde fixed tissue? They were processed routinely, 
starting in 10% NBF, with other tissues, and we are cutting them at 3u.
Thank you!
Sandy

Sandra J. Cheasty, HT (ASCP)
Histology & Necropsy Supervisor
UW-Madison, School of Veterinary Medicine


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[Histonet] Cut FFPE Slides for ISH

[Dorothy Hu via Histonet]

Hi Jennifer,

My ISH slides (FFPE) were stored in -20oC for several months or more than
one year and the targets were less abundant. I didn't use RNAscope though,
which is more sensitive. I don't know what kind of RNA your researcher try
to locate and need to do ISH within 24 hours after cutting?  Fresh cut
section will yield more signal for sure. I think as long as you have a few
copies of genes in your tissue, RNAscope should be able to detect.

Dorothy Hu
HSDM







> Message: 2
> Date: Mon, 28 Aug 2017 15:30:08 +
> From: "Jennifer  Phinney" 
> To: "Histonet@lists.utsouthwestern.edu"
> 
> Subject: [Histonet] Cut FFPE Slides for ISH
> Message-ID:
>  namprd05.prod.outlook.com>
>
> Content-Type: text/plain; charset="us-ascii"
>
> Hello Histonet,
> I am hoping someone can point me in the right direction to find some
> references for how long FFPE slides intended for ISH can be kept before
> staining.  I have a researcher who swears their slides have to be stained
> within 24 hours of being cut, which does not sound correct to me.  I've
> used ACD's RNAscope before and their protocol lists the timeframe as 3
> months (so this is a bit of a difference).
>
> There's a lot of information about fixation times, but I've come up empty
> handed for cut slides.
>
> Thanks everyone!
>
> Jennifer Phinney QIHCCM
> Project Coordinator
> Kansas State University
> Veterinary Diagnostic Lab
>
>
>
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Re: [Histonet] Paraformaldehyde Fixed Tissue

[Tina Van Meter via Histonet]

Hi Sandra,
What are the times for each processing station?​ Animal tissue is much
leaner, so it's easy to over process and become brittle to section.

Best,
Tina Van Meter




On Mon, Aug 28, 2017 at 11:30 AM, Sandra Cheasty via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi all,
> We are having difficulty sectioning mouse tissue, (brain,
> spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has
> anyone had issues with paraformaldehyde fixed tissue? They were processed
> routinely, starting in 10% NBF, with other tissues, and we are cutting them
> at 3u.
> Thank you!
> Sandy
>
> Sandra J. Cheasty, HT (ASCP)
> Histology & Necropsy Supervisor
> UW-Madison, School of Veterinary Medicine
>
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> Histonet mailing list
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>
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[Histonet] Cut FFPE Slides for ISH

[Jennifer Phinney via Histonet]

Hello Histonet,
I am hoping someone can point me in the right direction to find some references 
for how long FFPE slides intended for ISH can be kept before staining.  I have 
a researcher who swears their slides have to be stained within 24 hours of 
being cut, which does not sound correct to me.  I've used ACD's RNAscope before 
and their protocol lists the timeframe as 3 months (so this is a bit of a 
difference).

There's a lot of information about fixation times, but I've come up empty 
handed for cut slides.

Thanks everyone!

Jennifer Phinney QIHCCM
Project Coordinator
Kansas State University
Veterinary Diagnostic Lab
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[Histonet] Paraformaldehyde Fixed Tissue

[Sandra Cheasty via Histonet]

Hi all,
We are having difficulty sectioning mouse tissue, (brain, 
spinal cord, liver, and spleen), on paraformaldehyde fixed tissue. Has anyone 
had issues with paraformaldehyde fixed tissue? They were processed routinely, 
starting in 10% NBF, with other tissues, and we are cutting them at 3u.
Thank you!
Sandy

Sandra J. Cheasty, HT (ASCP)
Histology & Necropsy Supervisor
UW-Madison, School of Veterinary Medicine

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[Histonet] Cardboard File Drawers for Blocks

[Sandra Cheasty via Histonet]

Hello everyone,
Does anyone know of a company that sells the cardboard drawers 
and drawer dividers for filing blocks, separate from the outer cardboard file 
that the drawers slide into?
We came into possession of 25 outer files with no drawers, 
which means we need 200 drawers and dividers.
Thanks!
Sandy

Sandra J. Cheasty, HT (ASCP)
Histology & Necropsy Supervisor
UW-Madison, School of Veterinary Medicine

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