Re: [Histonet] paraffin embedding of frozen tissue

From:Gayle Callis

When working with mouse lung, it is best to fill the lung with OCT, then 
snap freeze.  OR fill the lung with 2.5 ml fixative, then cryopreserve with 
30% sucrose after fixation is finished, snap freeze.

We get perfect lung sections when we do this.

Filling is done - anesthetize the mouse or you can euthanize but no by 
cervical spinal cord dislocation.  Open chest and up under chin, expose 
trachea very carefully.  Make a tiny V shaped cut in TOP of trachea, then 
you can introduce an 19 guage dulled hypodermic needle attached to a 
syringe filled with either OCT or if you prefer fixative. Inject only 2.5 
ml (use a 3 ml syringe for this) if and watch the lung inflate.  AVOID 
overinflation of lung, it will blow alveoli away.  The OCT is NOT diluted, 
and whatever you do, do NOT severe the trachea or you cannot fill the lung 
- the trachea will retract into chest cavitity and you will fish around for 
it.  We use very sharp pointed curved cuticle scissors found in drug stores 
- these are stainless steel with the finest points and make excellent cut 
on top of trachea.

We clamp off the trachea where needle goes in using a mosquito hemostat, 
and carefully dissect the lung out, lifting carefully by trachea, remove 
heart, and snap freeze embedded in OCT (you can remove any lobe you wish at 
this point by laying filled lung on back of a Petri dish).

Some people perfuse the lung with fixative via heart, but you must severe 
the descending arteries behind the intestines - 18 guage or smaller needle 
with 10 ml syringe filled with fixative works.

At 02:33 PM 2/2/2006, you wrote:
>Greetings histonetters,
>I have recently found that sectioning frozen lung is a bit difficult. I 
>was wondering if  anyone has ever paraffin embedded previously fixed (and 
>cryproserved) frozen sections? And if  so what were the results? I am 
>performing immunohistochemistry / immunofluorecent staining on mice lung 
>tissue and I was just wondering if it was possible.  I also encourge 
>advice on the subject matter.
>I have learned quite a bit from this web site.
>Thank you very much.
>Carmen Contreras-Sesvold
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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