[Histonet] Processing adipose tissue

[Pheneger, Tracy]

Jerry-  Ispropanol is completely miscible in Paraffin and doesn't need
to be "cleared".  

David - I am also a proponent of Isopropanol processing of tissues, but
I don't usually have really fatty tissues (mostly I deal with animal
tissues).  Unfortunately, I don't think that the Isopropanol methodology
is infiltrative enough for really fatty tissues.  I worry that if you
increase your times and temps that you may damage the tissues.  You may
have to use an ETOH/Xylene - based system for these tissues.  

Please keep us informed on what you find out - there may be a paper or
poster for you!

Tracy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jerry
Ricks
Sent: Monday, January 30, 2012 5:15 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Processing adipose tissue


Hi David,

21 hours in isopropyl seems likw ea lot, and I don't see any "clearing
step to remove the IPA."

I've been using Slide Brite instead of Xylene, but I see that Rene Buesa
published a study indicating that mineral oil is the cat's meow for
xylene substitutes.

http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=10&ved=0CGM
QFjAJ&url=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%252
0as%2520xylene%2520substitute.pdf&ei=ny4nT73bM8axiQLk3dmQAQ&usg=AFQjCNFw
894if_bFAYgv6Uurw2MkO3Th5A



Jerry



> Date: Mon, 30 Jan 2012 12:20:24 -0600
> From: David.Burk <@t> pbrc.edu
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Processing adipose tissue
> 
> Esteemed experts,
> 
>  
> 
> We have many clients who want to process mouse and human adipose
tissue
> and are having some quality issues in the resultant slides.  
> 
> We have tried processing small chunks of tissue (<1 cm x 0.5 cm) on
our
> automated processor (Excelsior) as follows:
> 
> Tissue fixed for ~24 hrs in 10% NBF
> 
> 70% Isopropyl alcohol (IPA) for 3 hrs
> 
> 90% IPA, 3hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3 hrs
> 
> 100% IPA, 3hrs
> 
> Paraffin, 3 hrs
> 
> Paraffin, 3 hrs
> 
> Paraffin, 3 hrs
> 
>  
> 
> Embed and section at 5 um prior to H&E.  An example of what the
sections
> look like can be found here http://imgur.com/7RTGR .  
> 
> We also ran a sample on a "traditional overnight" EtOH/Xylene
processor
> (not at our facility) to compare results.  That image is here:
> http://imgur.com/GjJPg .  
> 
> What is obvious is that the membranes in the IPA processed tissue seem
> to "flap over" and don't look as crisp as the Xylene processed tissue.

> 
> We did notice structural defects in both samples (not shown) typically
> toward the middle of the specimens.
> 
>  
> 
> Does anyone know what is causing our IPA processed fat to have these
> "wide membrane" artifacts?  
> 
> We are going to repeat the process with an additional 30 minutes per
> step and raise the temperature of the steps to ~ 35 C.  
> 
> We are also going to cut the blocks at 2-3 um to see if it can reduce
> the appearance of the membranes.  
> 
>  
> 
> Thanks very much for any advice you may have for us.  We are pretty
> locked in to using xylene-free processing methodology if at all
possible
> but will entertain any suggestions you may have.  
> 
> If I can provide any further details about what we are doing on our
end,
> please let me know and I'll be happy to provide them.
> 
>  
> 
> Best,
> 
> David Burk 
> 
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