Re: Cryo-sectioning mouse lung

From:Gayle Callis

You need to fill the lungs with OCT, then section at -20C.  If you don't
fill the lungs, they will section like overhanging sand dunes, have lousy
air filled tissue that cuts horribly. 

I put out a protocol on Histonet recently on HOW to fill the lungs and
should should be in Histonet Archives. We do this all the time and no
longer need to rely on Cryojane for cryosectioning whole lungs, or even a
lobe.  We get good sections at 5 um.  The biggest danger is overfilling.
and blowing up alveolae.  A small immature mouse takes approx 1 1/2 mls
OCT, adult approx 2 ml, use a 3 ml syringe and fill with only 2 mls OCT,
this keeps you from overfilling the lung accidentally.  The lungs are
filled via a "v"shaped  cut in trachea, then insert a #19 gauge needle
(dulled) with OCT filled syringe.  You do NOT need to dilute the OCT.

Euthanize mouse with anesthetic overdose, open abdomen and detach the liver
which will severe all major blood vessels in abdomen i.e.
ascending/descending aorta to bleed out the mouse.  Open rib cage on sides
rather than midline (mouse should be pinned out at nose, legs, tail) and be
sure to cut through bone at top of rib cage near thymus/heart, and expose
trachea.  You want to see the whole lung with heart, thymus and trachea
exposed when you are done with dissection. Release trachea from surrounding
fascia gently, insert a small forceps or something small to elevate freed
trachea to raise it slightly, make a very tiny V shaped cut in TOP of
trachea.  If you cut across, trachea will recede into chest area and you
can't insert the dulled needle.  Insert needle into v cut, push gently,
slight distance towards the lung. The needle should still have some bevel
left on it, you should see the bevel facing up) inject OCT slowly, watch
lung inflate, use a mosquito hemostat to seal trachea (prevent leaking OCT)
slip needle out,lift up on lung, then dissect out lung, remove heart. Be
gentle.  The v shaped cut is made with a curved cuticle scissors from
Target or WalMart, these have the finest points, are German steel,
extremely sharp and dirt cheap.  

T  Once the syringe is inserted into the 

Embed in OCT, and snap freeze using a dry ice/isopentane slurry.   

If you continue to have problems - drive over to Bozeman - will be happy to
do hands on tutorial at Montana State University! 

 At 11:31 AM 7/8/2003 -0600, you wrote:
>First I would like to thank every one who responded to my inquiry about
>embryonic mouse spinal cord sectioning.  Now I need advice on
>Cryo-sectioning adult mouse lungs.  What tips do you all have?  What
>problems am I likely to run into?
>Dave
>David Walker
>MT(ASCP) MS Microbiology
>Staff Scientist
>CEHS/CSFNS/SPAHS
>University of Montana
>
>
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu





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