[Histonet] Processing adipose tissue

[David Burk]

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