[Histonet] Freezing mouse lungs

[Gayle Callis]

I recently put out an answer to your question, if not, here is the technic
I sent off today to a gentleman who had problems. 

As for mouse lung, you euthanize mouse with anesthetic as cervical
dislocation disrupts head and neck tissues needed for injection of OCT.
Open the abdomen, severe veins and arteries behind intestines to bleed out
animal, soak up blood with a PBS damp gauze.  Expose chest, open rib cage
and expose lungs, do not nick the lungs.  Expose trachea, but do NOT severe
the trachea. 
 
Free trachea from fascia gently with a fine tipped rounded forceps (eye
forceps) then using a cuticle scissor (extremely fine tipped, look for the
finest tip available) from WalMart or Target, place a tiny V shaped cut on
top of trachea BUT DO NOT CUT ACROSS TRACHEA OR IT RETRACTS INTO CHEST
AREA, ALL IS LOST!  Use an 18 gauge needle dulled with a sandpaper style
fingernail file, put it on a 5 ml syringe and fill syringe with OCT to 3 ml
or use a 3 ml syringe filled to 2.5 ml  (do not use any other cryomedia or
you will have grief).  Insert tip of needle gently into v shaped cut, push
toward lung a tidge, then inject OCT into lung, watching them fill until
just expanded, appear transluscent.  Clamp off trachea with a mosquito
forceps just below needle and as you pull out needle to prevent OCT
leakage.  DO NOT OVERFILL LUNGS with OCT OR you will blow up alveoli.
Younger animals require smaller gauge needles and less OCT - have several
needles dulled and ready to use if you can't get the needle inside the
trachea with ease. If you inflate with more than 3 ml OCT with any mouse,
you will damage lung.  

Dissect lung out with curved scissors, then remove heart with thymus.  Snap
freeze lung in Tissue Tek cryomold which we prefer for the thinner plastic.
Use a petri dish supported with a metal tube rack inside a styrofoam box,
and let dish FLOAT on Liquid nitrogen, do not put LiQ N2 in the dish, put
cryomold with tissue in dish, let block freeze.  Voila, perfect lung block
for frozen sections, no more lousy frozen sections and no freezing
artifact.  One can use a dry ice/2 methyl butane or hexane mixture, but dry
ice must be at top level of beaker and beaker surrounded with dry ice.
This is more toxic and dangerous with explosive solvent, and bad to breathe
- try the petri dish method.  You must NOT crack the OCT or lung with crack
too.

For paraffin work, we fix lungs in the same way by perfusing with NBF via
trachea.  

We used to use Cryojane, but this is far superior for our use these days.
Cryojane we reserve for undecalcifed bone frozen sections.  If the lung is
deflated which happens when you euthanize the mouse, the  Cryojane cannot
correct that and you still get funky collapsed lung frozen sections.  The
natural inflated lung morphology is retained with OCT filling.  We cut
these sections at -20C using high profile disposable blades.  

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)