[Histonet] H&E Protocol for Fresh-Frozen Brain and Spinal Cord

[Allyse Mazzarelli]

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