Detailed reply on how to Re: [Histonet] Frozen embedding of lungs

From:Gayle Callis

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.

Equipment needed

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 ( 
years later!)

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.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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