[Histonet] Processing adipose tissue

[Rene J Buesa]

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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