Detailed reply on how to Re: [Histonet] Frozen embedding of
gcallis <@t> montana.edu
Thu Mar 30 09:24:18 CST 2006
We fill the lungs with OCT (other cryoembedding medias may not work well if
they have only been tested on human tissues). We discovered this the hard
way, so test your cryomedia for murine work.
3 ml syringe with dulled 18 gauge needle. You do NOT want to have the sharp
cutting edges left on the bevel.
Fill syringe with OCT and make sure there are no bubbles in OCT.
Tissue Tek plastic embedding molds, dry ice/isopentane slurry for snap
freezing. NO CRYOSTAT freezing. If you want another snap freezing method
that eliminates the solvent, let me know, I will attach privately.
Cuticle scissors (stainless steel) found at drugstores, they are curved,
cheap and have the finest tips for what you are going to do. These are
excellent dissection tools for tiny murine tissues. Some type of blunt probe.
Mosquito hemostats with narrow, fine ends.
Euthanize the mouse with anesthetic overdose or CO2 gas since cervical
dislocation disrupts tissues and trachea.
Open abdomen up to lower jaw to expose lungs with heart attached by cutting
through the ribs and splaying these out so the lung and heart are visible
along with the trachea. Severe liver attachments from the lung cavity,
and bleed out the animal by severing ascending aorta and veins behind the
intestines, and use a PBS damp gauze sponge to soak up excess blood. Make
sure you DO NOT CUT THE TRACHEA nor disrupt it but gently expose it by
teasing away muscle and fascia around trachea. A blunt probe works very
well for this purpose. To minimize fur flying around, wet the animal with
PBS, not alcohol (that is a fixative) before opening the abdomen, pin down
legs, nose with hypodermic needles so body does not move around.
About half way down the trachea from the jaw area (better to be closer to
jaw than ribs), make a v shaped cut on the very top of the trachea but DO
NOT CUT across the trachea or the trachea retracts into lung cavity. Use
the fine tipped scissors to make the v shaped cut.
Insert 18 gauge needle into v shaped cut on top of the trachea. Keep the
needle flat and do not shove it in with force, you do not want to puncture
the trachea, and slide it so you can see the dulled bevel facing up but
into the lumen of the trachea. Inject OCT into the lung and watch it fill
but use only 2 to 2 1/2 mls or you will blow away the alveoli. You want
the lungs to be filled in the most lifelike form and size - not a tissue
Once you have filled the lung, take the mosquito hemostat, clamp off the
trachea below the needle bevel so you can gently lift and cut the
trachea. Gently dissect the lung with heart from pleural cavity - it will
come out intact using the hemostat to prevent OCT leakage from the lung
(backwash!). Using curved scissors, remove heart from the lung (unless you
want to see heart) embed the lung whole OR cut off the lobe you need, and
snap freeze immediately. Blocks can be stored at -80C for future dates (
We cryosection our OCT filled lungs at -20C, and never have problems with
morphology nor exploded alveoli. The latter is because care is take to NOT
overfill the lungs with OCT. The OCT does NOT have to be diluted, if it
is, it tends to leak or diffuse out from tissue. We have even used Thermo
Electrons green cryoembedding media which creates an interesting contrast
of lung tissue to the green goo but does not affect the sectioning qualities!
If you have any questions, contact me.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-4303 (FAX)
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