[Histonet] Mouse Muscle Fresh Frozen Sections H&E Stained, large spaces between fibers

[Donna J Emge]

I would be grateful to have some advice and detailed protocols from anyone
that works with frozen mouse muscle tissue. I am having a big problem with
the H&E stained 6 micron frozen sections of mouse muscle tissue. The fibers
have shrunk and there are large spaces between the fibers. The
Immunofluorescence  on another slide are fine. 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 shrank 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.

 

 

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