Hi Histonet! I was wondering if anyone could provide me with a reliable H&E protocol for fresh frozen tissue. Currently, we cut our slides on the cryostat and store them at either -20 or -80 (depending on the study director) until they require us to stain for morphology.
Typically the tissue isn't always perfectly snap frozen, and often times there are freezing artifacts. However, I would like to preserve as much as the morphology as possible without further damaging the tissue. I have really only had experience staining perfusion-fixed tissue using the following staining procedure: Water rinse Hematoxylin (7211 Richard Allen Scientific) for 30 seconds Water rinse under running tap Clarifier 1 (Richard Allen Scientific) with three quick dips Water rinse under running tap Bluing reagent (Richard Allen Scientfic) for 1 minute Water rinse under running tap 95% EtOH for 30 seconds Eosin-Y with Phyloxine (Richard Allen Scientific) for two quick dips 95% EtOH for 30 seconds 100% EtOH for 1 minute 100% EtOH for 1 minute 3, one-minute exchanges in Clear Rite 3 (a xylene substitute, Richard Allen Scientific) However, today I was asked to run an H&E on a fresh frozen slide. I post fixed in 4% PFA for 10 minutes immediately (not allowing the slide to thaw), and then ran the above procedure as normal. When I examined the slides under the scope, there appeared to be terrible freezing artifacts. The study director happened to think it was both a post-fix and staining issue, so I re-ran the procedure without post-fixing, (per his recommendation) as follows: Immediately into hematoxylin for 30 seconds Water rinse under running tap Eosin for 2 quick dips 95% EtOH for 1 minute 100% EtOH for 1 minute 100% EtOH for 1 minute 3, one-minute exchanges in Clear Rite 3 I saw slightly better morphology, but the director still was not happy with the outcome. I have spent the remainder of my afternoon researching different H&E protocols for fresh frozen tissue, and all seem to be relatively similar to the two mentioned prior. Does anyone out there in Histo-land consistently run H&E on fresh frozen tissue, specifically on slides that have been stored for a while? I work particularly with brain and spinal cord, but we occasionally will cut other tissue and organs. I would like to optimize this procedure as soon as possible. Thank you in advance! Regards, Allyse Mazzarelli _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet