[Histonet] RE: Processing adipose tissue

Tue Jan 31 16:16:38 CST 2012

Processing in IPA will give this picture. It is a lot gentler than ethanol/xylene.

I would also increase the fixation time (remember fat does not fix well, being hydrophobic). We have had good success with 10% formalin in alcohol.

I would not extend the processing steps at this time. I assume wax impregnation is at 60oC or more.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of David Burk
Sent: Tuesday, 31 January 2012 5:20 AM
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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