[Histonet] Cryomicrotomy on mouse lung - long reply

[Gayle Callis]

You wrote: 

I am looking for information relating to the preparation of inflated mouse
lung to be used for cryostat sectioning. Currently, the protocol being used
uses PBS to inflate the lungs which are subsequently tied off and embedded
in TBS or OCT and snap frozen in 2-methylbutane cooled in liquid nitrogen.
These lung samples need to be unfixed and are being sectioned at 8 microns
with disposable knives at a cryostat temperature of -32C.  Any warmer and
the TBS or OCT sections, but the lung doesn't. I am suspecting that the PBS
inflation may be playing into this - has anyone inflated with agarose,
sucrose (or something else) and sectioned successfully? Other ideas? With
appreciation...

Marilyn Wadsworth

Marilyn, 

You cannot inflate fresh murine lung with aqueous solutions, you end up
cutting ice.  Sucrose cryoprotection is commonly used for NBF prefixed
lungs - we use a simple OCT inflation of lung. 

Fill a 3 ml syringe with 2 1/2 mls OCT, on a dulled 18 guage needle - a
diameter that is perfect size for mouse trachea. Dull needle with a
fingernail file that has grinding sandpaper, we buy these that have 4
grits, are blue and pink in color at local WalMart.  You can leave a bit of
bevel on needle - inserts easier with bevel facing up, and you can see it.
We reuse these needles rather than reinventing the wheel.  

Euthanize mouse appropriately, using blunt/sharp scissors, open abdominal
cavity, and severe major blood vessels located next to spinal column to
bleed out mouse.  It is nicer to put a PBS dampened gauze to soak up blood.
 Detach liver, and open chest cavity carefully with blunt end of scissors
under ribs and do it cut lung. Do NOT remove heart at this time. Open rib
cage just about the trachea, do NOT cut the trachea, but you must cut
through bone above heart. Trachea is the route for inflating lung with OCT.
 Trachea is exposed, and free from surrounding fascia without cutting
trachea.  This can be done with a blunt probe or forceps that DOES NOT HAVE
A SHARP TIP, run it under trachea to free it from surrounding muscle, etc. 

You can raise the trachea using applicator sticks or a blunt probe, even
fine eye forceps,  mouse should be pinned firmly at nose, tail and legs (we
use syringe needles for pinning). Using an extremely fine tipped scissors
(we buy German steel cuticle scissors from Target or WalMart, they are
SUPERB and CHEAP with finer tips than vendors sell!), you cut a tiny "V"
shaped cut in TOP of trachea.  If you cut across it will retract into lung
area and you cannot capture it. Have a small lightweight fine tipped
mosquito hemostat forceps (SP?) ready for final dissection.  

Carefully, keep OCT filled syringe with dull needle flat, insert into v
shaped cut, watch it slide into trachea lumen with BEVEL SIDE UP, you can
push it towards lung but not too far,  then fill lung slowly with OCT. Use
a gentle touch, and do not force the needle or syringe plunger.  You do not
want to push needle through tracheal wall or into lung.  If you use more
than 2 1/2 mls OCT, you will blow the alveoli to bits, terrible sections.
To remove needle, slowly pull it out and quickly clamp trachea with
mosquito hemostat forceps just below tiny cut, keeps OCT from backwash,
maintains OCT in lung. Gently lift and using fine tip scissors finish
dissecting lung out, at this point, you can remove heart (know where it is
attached to not nick the filled lung).  

Embed lung in a Tissue Tek Cryomold with OCT,  large size mold, and snap
freeze with dry ice/isopentane slurry.  WE have had no success with any
other cryomold - Tissue Tek has thinner plastic excellent for snap
freezing. Holding one long tab with forceps, lower bottom of block into top
of dry ice/isopentane (make sure the dry ice level is high in a plastic
sample cup, work in a hood.  Watch the bottom begin to freeze, then let
mold/tissue/OCT sink into slurry. It takes approx 10 seconds to freeze.
Liq Nitrogen cooled isopentane will crack OCT/block, too cold.  Remove and
let isopentane evaporate, mount block on chuck, and cut at -20C, colder is
not necessary with OCT filled lung, in fact, creates crunchy sections if
TOO cold.  We cut at 5 um, no problems and have perfect sections on whole
OCT filled lung. We use disposable high profile blades, Accuedge are a
preference, and brush technic - antiroll device resides in a drawer.   

To get away from isopentane, float a plastic petri dish on liquid nitrogen
using a platform in N2 to support dish,  but do not let N2 go into dish, it
must float on N2 and surround dish.  Set embedded lung in dish and let it
freeze, no artifact, perfect sections. 

If you need help with snap freezing setup, I will discuss that privately.
We trim plastic edges from 3 sides of cryomold so we can freeze faster and
store blocks in 50 ml centrifuge tubes at -80C.  

There are other snap freezing methods that work, but liquid N2/isopentane
requires too much recooling when you have 20 mice lined up for dissection,
etc. We never use this method for a large experimental tissue collection. 

  

     

 





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 <@t> montana.edu