Re: mouse lung for frozens and paraffin - long reply on technic

From:Susan Bell


I tried the trachea nicking technique Thursday and love it.  Much 
better than cutting the trachea and then trying to feed the needle 
into the end.  I still use the feeding needle to inject the formalin. 
I think that is my preference.

Question:  What do you anesthetized with?  We almost always have to 
collect blood, so the PBS perfusion to get rid of blood is not 
something I'm likely to do often, but I am curious about it, just in 
case.  We use isoflurane which slows the heart down so much it's 
sometimes hard to get much blood!

Susan Bell
Research Assistant
Targeted Genetics Corporation
1100 Olive Way  Suite 100
Seattle WA  98101
Phone:  206-521-4830
Fax:  206-223-0288

At 11:34 AM -0600 9/4/02, Gayle Callis wrote:
>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)


Susan Bell
Research Assistant
Targeted Genetics Corporation
1100 Olive Way  Suite 100
Seattle WA  98101
Phone:  206-521-4830
Fax:  206-223-0288

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