[Histonet] Processing adipose tissue

Tue Jan 31 08:57:11 CST 2012

David:
21 hours dehydration is too much, but the main problem resides in the "abrupt" transition from alcohol to paraffin wax, especially with fat tissue.
Under separate cover I am sending you the article I referred to.
René J.

--- On Tue, 1/31/12, David Burk <David.Burk <@t> pbrc.edu> wrote:

From: David Burk <David.Burk <@t> pbrc.edu>
Subject: RE: [Histonet] Processing adipose tissue
To: histonet <@t> lists.utsouthwestern.edu
Date: Tuesday, January 31, 2012, 9:51 AM

René,
Thanks very much for your input on this matter.  I also want to go ahead and thank the other two (Jerry and Karen) who have chimed in on my question.  René, could you please elaborate on "the problem resides in the dehydration time"?  Am I over doing it with 21 hours of IPA?  As for the suggestion about introducing an intermediate step, I am going to find out if that is doable on our Excelsior and, if so, replace the last (9th) IPA step with either an IPA/Paraffin mix or perhaps a mineral oil mix.

If anyone else has further suggestions as to help us get nice crisp membrane structure from our adipose sections I will be very happy to hear them.  Also, if you need additional example images from our sectioned material, I can provide them easily.

Best,
David B.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, January 31, 2012 8:34 AM
To: histonet <@t> lists.utsouthwestern.edu; Jerry Ricks
Subject: RE: [Histonet] Processing adipose tissue

I agree that 21 hours is too much, but using isopropyl alcohol does not a "clearing" step because isopropyl alcohol will dissolve paraffin wax at 50ºC and above.
This is the method I have published else were with the intermediate step of mineral oil (which is paraffin of low molecular weight) to reduce the gradient and protect the tissue structure.
The Peloris processor routinely uses this sequence (isopropyl → paraffin wax) and is widely used in Australia.
The problem resides in the dehydration time. I will also suggest an intermediate step of isopropyl alcohol+paraffin wax 1:1 between the last alcohol and the first paraffin wax bath.
René J.

--- On Mon, 1/30/12, Jerry Ricks <rosenfeldtek <@t> hotmail.com> wrote:

From: Jerry Ricks <rosenfeldtek <@t> hotmail.com>
Subject: RE: [Histonet] Processing adipose tissue
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, January 30, 2012, 7:15 PM

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=0CGMQFjAJ&url=http%3A%2F%2Fwww.ebsciences.com%2Fpapers%2FMineral%2520oil%2520as%2520xylene%2520substitute.pdf&ei=ny4nT73bM8axiQLk3dmQAQ&usg=AFQjCNFw894if_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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