Hi Dane, Fading and especially leaching of the eosin, will occur if slides are incompletely dehydrated. Flip out the subsidiary condenser lens (or drop the condenser) and look for refractile water droplets. Certain mountants can also cause fading of H&Es. I would also be concerned about the xylene substitute.
Alternatively, after staining and ethanol dehydration, air dry the slides (making sure they are completely dry) and directly coverslip with your mountant (without the substitute added). This will reduce the risk of xylene exposure. Hopefully this is useful Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of Medicine, University of Western Sydney Tel: 612 9845 3306 Fax: 612 9845 3318 Pathology Department the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: Hill, Dane via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Tuesday, 17 March 2020 1:01 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Frozen section slides fading Hello all, Thank you, in advance, for your help and for the information you've already posted online. It is helpful. I am hoping you can help me troubleshoot an issue we are having. I am the Mohs fellow at Texas Tech dermatology. For the last 9 months or so we have been noticing that the longer our frozen section slides are away from the day of surgery, the more they develop these pink, amorphous spots in certain areas of the tissue. The further away from the surgery, the more faded they get. The actual tissue remains intact on the slide, with complete epidermis evaluable, but it's almost as if all the hematoxylin fades or something because it just turns into that bright pink color, but does not hold any of the purple. We have tried changing our xylene substitutes and even changed the hematoxylin and eosin stains. The thinner (5 micron cuts) are affected long before the 20 micron cuts. Photos were too big to be attached, but I can PM someone with them, if needed. They are from slides prepared just a few weeks ago and are just starting to fade. In about 6 months they will turn all pink. Interestingly, this is not happening on any of our permanent sections. They remain perfectly stained for years. And it will happen, even if we don't "bake" any of the slides or heat them in storage. The only thing the techs say they do differently for the frozen sections is use xylene substitute in place of xylene. They also mix a little xylene substitute in the glue to cover slip the slides. I wasn't sure if that could be contributing. If there are any testing protocols of specific reagents we use or adjustments in staining that have previously remedied the issue, I'd be very grateful as we have been unsuccessful at pinning it down. Thank you, again! Dane Hill _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This message is intended for the addressee named and may contain confidential information. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message are those of the individual sender, and are not necessarily the views of NSW Health or any of its entities. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
