mouse lung for frozens and paraffin - long reply on technic

From:Gayle Callis

We fill (not really a perfusion, which implies solution flowing into and
through organs) the lungs for both frozen or paraffin work. 

For both technics, we open mouse abodominal/chest cavities, sever major
arteries behind intestines to "bleed out" animal.  A PBS dampened gauze is
inserted to soak up blood for less mess. Make sure the liver and
pericardium surrounding lung and heart are freed for easier removal of
heart and lung after filling, and open  
ribcage totally with trachea gently exposed and leading into lung.  Trachea
must be freed from surrounding fascia/muscles.  

One can slightly raise trachea with with a fine forceps or applicator
stick, pipettor tip, etc to put a bit of tension on trachea itself - this
can make the next step easier.  Make a small v shaped nick on top of
exposed trachea using a very fine dissection scissors (I like very tiny,
sharp cuticle scissors purchased from Target or Wal Mart! Cheaper too!).
Do not make this v shaped cut too close to lung and NEVER cut across
trachea totally, or it will retract towards lung, and you cannot find tiny
end of trachea for next step - the needle insertion.   You will now have a
hole in top of trachea - THE most important part of this technic.  
For frozen sections, use a syringe (3 ml size only, larger syringes are
harder to handle with tiny mouse) with NO MORE than 2 mls OCT (it does NOT
have to be diluted), use a #18 gauge needle that is dulled to remove sharp
edges but level bevel intact. Don't cut off end of needle! #18 gauge is a
perfect match of mouse trachea lumen diameter. If the animal is immature,
try 1.5 mls or even 1 ml of OCT and possibly a smaller gauge needle.  The
needle should fit snugly without tearing during entry into trachea. Oral
gavage needles can be used, but the bulb on end is a bit more difficult to
insert and leakage tends to be worse during filling. 

Sandpaper is used to dull needle, but polish it with fine paper to remove
rough edges.  A slight bevel left on this dulled needle is helpful during
insertion.   Insert dull needle with bevel facing up into tiny hole of
trachea using a flat angle approach to go down trachea - towards lung.
With care, fill the lung SLOWLY with OCT and watch lung inflate.  If you
use more than 2 mls OCT, you can "blow up" lung alveoli, to little OCT
results in partial collapse, poor sectioning.  Some people dilute the OCT
1:1 with PBS, but this leaks out rapidly, we find undiluted OCT is fine or
try a lesser dilution.   You could also dilute OCT with 20% sucrose, but
not needed since OCT means Optimal Controlled Temperature, and fills air
spaces with media suitable for cryomicrotomy, and basically cryoprotects
with good contacts inside alveoli.    

After filling lung, pull needle out quickly,  clamp trachea with mosquito
hemostats to prevent OCT leakage from hole in trachea.  Using hemostats,
lift trachea carefully, and dissect lung out of thoracic cavity. We remove
heart at this time. Embed whole lung or a selected lobe into OCT in a
Tissue Tek disposable mold, snap freeze.  We have several ways to snap
freeze, one with dry ice isopentane slurry or a liquid nitrogen method free
of precooled isopentane. After filling with OCT, one can lay a lobe on top
of petri dish, slice it with a scalpel blade for a bisected lobe to see
more bronchioles.  

For whole mount beta gal staining of lungs, the lungs are filled with
paraformaldehyde fixative the same way, dropped into fixative overnight,
followed by  whole mount staining and at completion, lungs are allowed to
cryoprotect with 20% sucrose in buffer- overnight or fixative for long term
storage.  Sucrose protected lung is snap frozen, and sectioned at -27C or
so, colder has proven better for sucrose protected lungs, it oozes out at
-20C! Messy and sticky!    

For paraffin sections, lungs are filled with NBF or cold paraformaldehyde
using the same inter-tracheal technic, heart is removed, filled lung is
immersed into fixative for longer fixation, then processed whole.  Care is
taken to never cut trachea too short IF this tissue must be seen, and best
if left a bit longer for handling purposes.  Since we have filled lungs
with fixative, the morphology has been superior, undamaged - the same for
frozens/OCT filled lung.   

One lab here performs a perfusion to remove blood. Mouse is anesthetized,
chest cavity opened, arteries severed, and while heart continues to beat,
10 - 15 mls PBS in injected into heart chamber (lower left side) to permit
free flow through heart.  As PBS is injected slowly into heart, it flows
through heart into lungs and out severed arteries.  The heart actually
turns white! as do the lungs  - indicating blood being flushed out of these
organs.  After PBS perfusion, a syringe with NBF is attached and lung,
heart is perfused with fixative, immersed, processed. etc.   Their murine
lung work is without compare! They get perfect sections for both paraffin
or frozens - a never fail situation.  

For frozen section work, we formerly used Cryojane for air filled lungs.
Since we perfected this filling methods, we obtain perfect frozen sections
with OCT support of lung tissue internally and externally on all age mice.  

Filling lungs takes practice, you need to have a steady hand, patience, and
learn to think small - the mouse is not easy to deal with for perfusion,
infusion ie filling of lung? Buy good, mini-dissection tools.  We use
ARISTA company out of New York for inexpensive, discounted tools that are
excellent.  Dull scissors will always be the enemy, keep a good supply and
learn to not abuse them.   

Formalin or paraformaldehyde fixation - even PLP fixed frozen sections, has
resulted in too much autofluorescence, I agree another fixative may be
superior to avoid this problem or use a fluorochrome that is a different,
but very bright color as contrast to autofluorescence.          

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

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


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