|From:||Kim Kusser <firstname.lastname@example.org>|
We infect mice with Flu and pneumocystis, and this is how we collect lung tissue.
1. Bleed mouse out while heart is still pumping
2. Fill lungs with OCT (we do it with 100% OCT) by cutting a small slice in the trachea,
insert needle (we grind the points off our needles), then slowly push in ~1ml of OCT or
enough til all lobes are inflated
3. Then take what you want, put in containers filled with OCT, and freeze
(Note: We freeze by putting petri dishes on liquid nitrogen, and placing the containers with the OCT/tissue on that).
I cut my frozen lung sections at ~ -22-25c and 7microns. I then label them enzymatically or with fluorescence.
Hope this helps
I would be very grateful for some advice on the following:
We are interested in doing histology and laser capture microdisseciton
(LCM) on mouse lungs. LCM allows one to dissect specific cell populations
from the tissue and subsequently measure mRNA in these cells. This requires
that the tissue be frozen and not fixed in formalin. We also want to
distend the lungs before freezing so that the architecture is preserved as
well as possible. We have previously done this with formalin, injecting it
through the trachea. A search through the archives suggested that we dilute
OCT 1:1 with PBS and use this to distend the lungs before snap-freezing
them. Is this what would be recommended by most people? Would it be better
to use sucrose with OCT to ensure further cryoprotectant effect?
A question - apparently ethanol/acetone is not damaging to mRNA. Would it
be feasible to distend the lungs with one of these fixatives. I realise
that they have poor penetration, but would it be adequate for fixing the
airways and alveoli of a mouse? If this worked, presumably one would not
have freeze the lungs, except when cutting the sections.
I would be very grateful for any advice
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