air filling Re: [Histonet] Frozen embedding of lungs

[Gayle Callis]

More on snap freezing murine lungs. and long discussion.

Air filling a lung is not going to help with support during 
cryomicrotomy.  The OCT must be in close contact with inner lung surfaces 
i.e replace the air, to ensure lung cryosections correctly.  If not, air 
filled or partially air filled lungs (normally what you end up after a 
dissection and snap freezing,  and what Kim was experiencing) still 
cryosections like a crumbling, overhanging sand dune.  Air filling is 
really no different than taking a lung from an animal who has breathed and 
although the lung is collapsed enough air remains to give the crumbling 
section syndrome even after proper snap freezing.  We have experienced this 
too many times until we performed OCT filling of the lung to replace the 
air.   Lung morphology in our hands is intact and excellent.

Some individuals fill or perfuse a lung with formalin or paraformaldehyde. 
Fixed lungs should be cryoprotected with 30% sucrose to replace the water 
(in the fixative ) to prevent large water ice crystal formation.  Slow 
freezing creates the same problem by large water ice crystal formation in 
ANY fresh tissue.  Water seems to be the enemy here.

It is very important to snap freeze OCT filled then OCT surrounded/embedded 
tissue with a proper snap freezing setup as John described and never use a 
cryostat freezing apparatus that cannot achieve a cold enough 
temperature.   We embed a whole lung in large plastic cryomolds, then snap 
freeze in a dry ice/isopentane or hexane mixture  at approx -90C to have 
complete snap freezing of OCT filled lung  in about 10 seconds or 
so.  Liquid nitrogen cooled isopentane is just another method.  That rapid 
freezing is critical for prevention of freezing artifact.

  One could choose to freeze the OCT filled lung without embedding, but 
Dodds taught us to coat fresh bone with a water soluble 70,000 PVA before 
immersing into a dry ice/hexane slurry. The MW weight of this PVA means the 
PVA molecule is too large for proper infiltration into tissue 
micro-spaces.  Our Center for Biofilm Engineering indicated the PVA should 
be MW of 40,000 or less for infiltration purposes.

For the tissue coating method, we dilute OCT (since it already contains 
PVA) with PBS (1:1) and dip the fresh tissue, OCT filled lung, or bone in 
this mixture prior to snap freezing.  It could be assumed this thin OCT 
layer protects the tissue during -80C storage and maybe from direct contact 
with the hexane or isopentane although these may not affect the tissue in 
any way.

OCT bottle has all ingredients stated clearly.  OCT contains 10.24% w/w 
polyvinyl alcohol ,4.26% polyethylene glycol (the MW of PVA and PEG is not 
given for proprietary reasons) and 85.50% non-reacative ingredients which 
could be water.  Dr. McCormick developed OCT for Tissue Tek many years ago 
and per private conversation he said could vary the ingredients for any 
given cryotomy temperature desired - very clever!    The lung is NOT 
cryoprotected with OCT as that is not the purpose of this embedding 
media.  In order to cryoprotect, an ingredient i.e.30% sucrose, should 
infiltrate into tissue micro-spaces to replace water. The MW of the PVA and 
PEG in OCT may be too great to achieve proper infiltration into tiny tissue 
spaces., hence the word 'surround'.   It has been observed that some say 
they "infiltrate" with OCT by letting tissue sit overnight - and it may 
actually be more the OCT oozes into the irregular tissue spaces unless the 
space is big enough to let a large molecule in?   Some mix OCT with 20% 
sucrose (1:1 mixture) and let this sit.  The sucrose is probably what 
infiltrates the tissue spaces to replace water and NOT the PVA of PEG of 
the OCT.

It would be an interesting experiment to take Thermo Electrons colored 
cryoembedding media and see if there is any infiltration into the tissue 
spaces - a line of green or blue in the tissue?

  The OCT bottle says this media is to "bind tissue to the specimen block 
and to surround and cover the tissue specimen".  I interpret this as 
meaning NOT an infiltration medium like hot paraffin,  but only as a means 
of supporting (air really doesn't do this very well) by direct contact with 
tissue surfaces for cryomicrotomy at optimal cutting temperature (we use it 
in a temperature range from -15C to -35C.  If my thoughts are incorrect, 
hopefully Dr. McCormick is looking in and will make further comments.

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717




  At 09:47 PM 3/29/2006, you wrote:
>Why not inflate with air (Pasteur pipette in
>trachea) and then plunge into isopentane (cooled
>by liquid nitrogen) or acetone (with dry ice ion
>it). Neither is ideal, but both freezing methods
>protect quite well against ice crystal damage; the
>crystal holes are much smaller than cells.
>
>Coating a specimen with goop before freezing
>ensures that the cells of the specimen are the
>last and most slowly frozen things in the blob.
>That makes for maximum damage by ice crystal
>formation. With slow freezing most of the the ice
>crystal holes are about the same size as a cell,
>and the tissue architecture is 100% wrecked.
>Unfixed objects must be frozen alost instantly. A
>blob of hydrophilic polymer can be added
>afterwards to protect against freeze-drying of
>stored specimen. OCT (whatever it is; ?polyvinyl
>alcohol) is a physical support for specimens being
>sectioned with a cryostat or an olde-worlde
>freezing microtome. It does not infiltrate the
>tissue in the way paraffin wax enters every
>dehydrated crevice. Most of the functions of OCT
>are duplicated by other  hydrohilic polymers such
>as gelatin or agar.
>
>The abbreviation OCT is for Optimum Cutting
>Temperature. That has nothing to do with
>protection against freezing artifacts. It relates
>to the hardness of the polymer at some temperature
>that was, many years ago, considered optimal for
>cutting frozen sections.
>
>John Kiernan
>London, Canada
>-----------------------
>
>Kim O'Sullivan wrote:
>>Hi all,
>>Does any one out there have a successful method for inflating mouse lungs 
>>to freeze. We currently freeze them unfixed in OCT, but of course the 
>>morphology is shocking as the lungs have collapsed. Is there a method 
>>that preserves the morphololgy without collapsing or imploding the lungs.
>>Any advice will be appreciated
>>Kim O'Sullivan
>>Centre for Inflammatory Diseases
>>Department of Medicine
>>Monash University
>>Melbourne
>>Australia