[Histonet] Cryomicrotomy on mouse lung - long reply
gcallis <@t> montana.edu
Tue Aug 26 17:32:13 CDT 2003
Yes, we have diluted OCT with PBS 1:1, but found it was not necessary. Lung
filled nicely plus diluted OCT is more aqueous, looks funny at times when
frozen as water concentration increases. If it was any more dilute,
sectioning was poorer. We had good sectioning with 1:1 diluted OCT, but it
also tends to diffuse out of lung - I had to work quickly. We don't use TBS
embedding media (if it isn't broken, don't fix it attitude) and are finding
some brands don't do the job as well as OCT, but that is a preference plus
application usage (filling small intestine lumen with OCT is also done
after rinsing feces out with PBS, sort of the acid test of cryoembedding
media for us).
People really need to try different cryomedias, note plural. We discovered
some do not hold tissue as well resulting in sections compressed or
cryoembedding media pulling away from villi inside murine intestine. It
pays to try them all just to see if one works better with rodents, animal
tissues. Many of these media are developed and tested in clinical/human
situations and then the "mousie" types come along and do something entirely
different, testing cryoembedding media to their limits. This doesn't mean
a product is inferior, just not best for us.
I have tried Shandons colored media in lung and it was so neat to see a
colored goo inflate the lung i.e. green or blue, theirs also worked well
with lung. Not tried Shandon's with intestine though. So advice is if you
get poor sections with one cryomedia, try others - vendors graciously send
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
At 07:40 AM 8/27/2003 +1000, you wrote:
>Have you tried diluting the TBS or OCT mountant?
>P.O. Box 533
>Wentworthville NSW 2145
>Ph 02 9845 7774
>----- Original Message -----
>From: "Gayle Callis" <gcallis <@t> montana.edu>
>To: <Histonet <@t> lists.utsouthwestern.edu>
>Sent: Wednesday, August 27, 2003 4:19 AM
>Subject: [Histonet] Cryomicrotomy on mouse lung - long reply
>> 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
>> 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
>> inflation may be playing into this - has anyone inflated with agarose,
>> sucrose (or something else) and sectioned successfully? Other ideas? With
>> Marilyn Wadsworth
>> 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
>> 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
>> 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
>> Trachea is exposed, and free from surrounding fascia without cutting
>> trachea. This can be done with a blunt probe or forceps that DOES NOT
>> 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
>> 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
>> 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
>> 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,
>> 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
>> 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
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
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