[Histonet] Shrunken Fibers Problem Mouse Muscle Fresh Frozen Sections H&E Stained

[Donna J Emge]

I am having a big problem with the H&E stained 6 micron frozen sections of
mouse muscle tissue. The Immunofluorescence of adjacent sections on another
slide are fine, but on the H&E stained slides the fibers have shrunk and
there are large spaces between the fibers. The sections are from the same
tissue, adjacent slides, cut at the same time. The sections are from fresh
muscle tissue sticking out of tragacanth paste that was flash frozen in a
beaker of isopentane in liquid nitrogen. The sections on slides were brought
to the lab on dry ice and stored in our -20 for H&E staining the next day.
The sections looked fine under the scope unstained, but shrunk and had large
spaces after H&E staining. I did not see freezing artifact: holes, spidery
blown out cells etc on the unstained slides just before staining and as I
said the IF adjacent slides of the same tissue looks fine. I believe the
tissue is reacting with the alcohol and xylene since for IF only water based
reagents are used. 

 

Stain protocol: Air dry and bring sections to RT, 10% NBF 5min, 95% Etoh
1min, 80% Etoh 1 min, H2O 2min, Harris Hematoxylin 2 mins, H20 1min, 0.5%
HCl 70% Etoh 2 secs, H20 2mins, Sat. Lithium Carbonate blueing 30secs, H20
1min, 80% Etoh, Eosin, - dehydrate, clear, mount w/ xlyene based mount.

 

All other frozen section tissue types look great stained with this protocol.
The H&E actually looked great, just not the muscle section morphology after
H&E staining. I see Google images of frozen muscle sections stained with H&E
so there must be a way to do the stain without this effect on frozen muscle
sections.

 

I would be grateful to have anyone that does work with frozen mouse muscle
tissue give me some advice and detailed protocols. 

 

Thank you,

Donna

 

Donna J. Emge, ASCP-HT

Mouse Histology and Phenotyping Laboratory Manager

Northwestern University

Olson Pavilion 8-333

710 North Fairbanks Court

Chicago, IL  60611

d-emge <@t> northwestern.edu

312-503-2679