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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of David Burk Sent: Tuesday, 31 January 2012 5:20 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************************* _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
